Protein Sorting and Vesicular Traffic in the Golgi Apparatus

Protein sorting and vesicular traffic in the Golgi apparatus

M.G. Farquhar1 and H.-P. Hauri2

JDivision ofCellular and Molecular Medicine and Department ofPathology, University ofCalifornia, San Diego, CA 92093-0651, USA 2Department ofPharmacology, Biozelltrum, University ofBasel, CH-4056 Basel, Switzerland

Summary 65 Introduction 65 Major routes of protein and membrane traffic to and through the GA 66 Overview oftraffic along the exocytic pathway 66 Models ofGolgi organization 68 Mechanisms of sorting, targeting and transport from the ER 71 Exitfrom the ER occurs at specific export sites 71 Selective transportfrom the ER vs bulk-jlow models 72 Transit through the ER-Golgi intermediate compartment (ERGIC) 74 Other proximal pre-Golgi, pre-ERGIC, smooth ER compartments 76 Mechanisms for retention and retrieval ofresident ER proteins: Current models 79 Transport, processing and sorting by the GA 80 Contributions ofin vitro assays 82 The SNARE hypothesis 82 Golgi compartments and post-translational processing oftransported proteins 83 Golgi-specific functions . 83 How many Golgi compartments are there? 83 Proposed mechanisms for retention and retrieval ofresident Golgi proteins 86 Transmembrane domain retention signals 88 Formation ofinsoluble aggregates too large to enter transport vesicles 88 The kin recognition model 89 Bilayer-mediated sorting 89 Sorting at the TGN 90 Sorting oflysosomal enzymes 92 64 M.G. Farquhar and H.-P. Hauri

Sorting and packaging ofsecretory proteins into regulated secretory granules 92 Constitutive secretory vesicles: A default pathway? . 93 Sorting in polarized epithelial cells 94 Mechanism of vesicle budding, docking and fusion 95 Different transport vesicles - Common principles 95 Coat proteins and vesicle budding 97 Clathrin/adaptin-coated vesicles (CLA-CVs) 97 COP I-coated vesicles 99 COP II-coated vesicles (COP II-CVs) 102 Vesicle docking andfusion 104 Less well-characterized transport vesicles 108 Constitutive exocytic transportfrom the TGN to the plasma membrane .. 108 Transport from the TGN to the apical and basolateral domains ofthe plasma membrane in polarized epithelial cells 108 Vesicles involved in the recycling ofmannose 6-phosphate receptors (MPRs) from the late endosome to the TGN .. 108 Vesicles that mediate recycling ofplasma membrane proteins from early endosomes to the plasma membrane 109 Caveolae 109 Regulation ofmembrane traffic by small GTPases, heterotrimeric GTPases, and phosphoinositides 110 Small GTPases 110 Heterotrimeric G proteins 113 Phosphoinositides 114 Conclusions . 115 Acknowledgments 116 References 116 Protein sorting and vesicular traffic in the Golgi apparatus 65

Summary. During the last ten to 15 years there has been an explosion of new infonnation on mechanisms of protein sorting and vesicular traffic along the exocytic pathway, that has allowed us to extend and modify earlier models. We now know that newly synthesized proteins translocated to the endoplasmatic reticulum (ER) are not automatically transported to the Golgi stack. They must pass through an elaborate ER quality control system which assures that they are properly folded and assembled before leaving the ER. We also have learned that resident ER proteins are nonnally retained in the ER by mechanisms that are still poorly understood, but if they happen to escape they are retrieved by binding to receptors located in the cis-Golgi and transported back to the ER. A new compartment, the ER to Golgi intennediate compartment (ERGIC), with distinct morphological and biochemical features, has been discovered. In mammalian cells cargo is shuttled from the ER to ERGIC via one type of vesicle (COP II-coated vesicles) and from ERGIC to the cis-Golgi network via another (presumably COP I-coated vesicles). It has long been postulated that cargo exits the ER at specific sites, the transitional ER elements, and that transport is nonselective and occurs by so-called "bulk-flow". However, there is growing evi• dence that proteins are sorted for packaging into carrier vesicles rather than being transported by bulk-flow. Cargo undergoes concentration (five to ten times) at the time of exit. The mechanism by which cargo is transported through the GA from cis to trans remains to be elucidated. According to one model it is mediated by vesicular transport. Another view is that transport my occur by cisternal progression whereby the cis-cisterna gradually matures to become the transmost cisterna. Cargo is modified in transit by Golgi-specific post-translational modifications (glycosylation, phosphorylation, sulfation, etc.), but no further concentration takes place until proteins reach the trans cisternae and TGN. There, newly synthesized proteins are selectively packaged into at least four different containers - i.e. secretory granules, two types of constitutive secretory vesicles (apical and basolateral cognate), and clathrin-coated vesicles carrying lysosomal enzymes. The mechanisms of sorting are best understood in the case of lysosomal enzymes sorted via mannose 6-phosphate receptors. Sorting into regulated granules is believed to occur by aggregation, and sorting of at least some proteins into constitutive vesicles by tyrosine-based signals. The mechanisms by which resident Golgi proteins are retained in the Golgi stacks are still poorly understood, but there is a general consensus that important infonnation resides in the transmembrane domain of integral membrane proteins. During the last few years infonnation derived from the study of Golgi transport in vitro, yeast sec mutants, and synaptic vesicles has converged and demonstrated that vesicles involved in transport at different steps along the exocytic pathway have common features. Most have prominent coats of which there are several types. It is thought that vesicle budding is initiated by the assembly of the protein coat, thus triggering assembly of elaborate membrane and cytosolic protein complexes, unique for each vesicle population, that drive vesicle budding and fission. Included in the fonning vesicle are cargo proteins, cargo receptors and membrane proteins required for vesicle docking. Pairs of integral vesicle and target membrane proteins (v-SNARES and t-SNARES), with specific family members assigned to different stations, assure docking to the appropriate membrane receptor. Soluble cytosolic proteins (NSF and SNAPs) serve as common fusion machinery for different vesicle relays. An exception is represented by the vesicles involved in apical delivery of proteins in polarized secretory cells where glycolipids and glycans appear to be involved. Attention is currently focused on how traffic is regulated and how signals are transduced from one cell compartment to the other. Small GTPases of the Rab and ARF families, heterotrimeric G proteins, and phosphoinositides are known to be involved, but understanding of their specific roles is still fragmentary.

Introduction

IfCamillo Golgi were alive today we can imagine his surprise and delight to learn of the frenzy of research activity focused on the organelle that bears his name and the demonstration of the central role it plays in so many important cell processes. We have known and understood for some time that the Golgi apparatus (GA) functions as a major biosynthetic organelle involved in the pack• aging and post-translational modification of newly synthesized proteins. More recently we have learned that the GA serves as the cell's main sorting and distribution station for protein and vesicle traffic. In this review we will outline recent progress in understanding the role of the GA in protein sorting, protein distribution, and vesicular trafficking. We will focus on findings obtained and con• cepts developed in the last ten to 15 years. Earlier work and the historical background are covered 66 M.G. Farquhar and H.-P. Hauri in the chapter by Berger and have also been summarized in previous reviews (Farquhar and Palade, 1981; Farquhar, 1985). For different perspectives on sorting and trafficking by the GA the interested reader can also refer to several other excellent recent reviews (Harter and Wieland, 1995; Harter et aI., 1996; Hong, 1996; Pfeffer, 1996; Hauri and Schweizer, 1997). We will divide our synopsis into four parts - in the first part we give an overview of the route followed by proteins in transit along the exocytic pathway through Golgi and pre-Golgi compartments, in the second and third parts we summarize what is known about the sorting, targeting and transport of proteins by the ER and GA, and in the fourth part we summarize recent progress in understanding the molecular mechanisms of vesicular transport between the GA and other cell compartments.

Major routes of protein and membrane traffic to and through the GA

Overview oftraffic along the exocytic pathway

It has been known since the classical work of Palade and coworkers (Palade, 1975) that secretory proteins move vectorially through the cell- i.e. from the ER, to transitional elements of the ER, to the GA where they are packaged into granules or vesicles (Figs I to 3). It is now clear that all secretory or membrane proteins, with the exception of resident ER proteins, bearing a leader or signal sequence are translocated into the ER lumen or inserted into the ER membrane and follow a similar route. A typical secretory or membrane glycoprotein, for example, is targeted to the ER by a hydrophobic signal sequence which interacts with the signal recognition particle (SRP) complex, and is translocated across the ER membrane to the ER lumen or inserted into the ER membrane where it is glycosylated and undergoes folding and, in the case of oligomeric proteins, assembly into homo- or hetero-oligomers. Improperly folded proteins are retained in the ER through interaction with various chaperones such as BiP and calnexin assuring proper "quality control" over protein export (Hurtiey and Helenius, 1989; Gething and Sambrook, 1992; Helenius et aI., 1992; Bergeron et aI., 1994; Helenius, 1994). Once the protein is "mature" it exits the ER via coated vesicles that bud from transitional elements of the ER. Recent evidence indicates that transport of secretory as well as membrane proteins is selective and that products are concentrated upon export from the ER. The newly synthesized proteins are then transported sequentially through the ER-Golgi intermediate compartment (ERGIC) (Itin et aI., 1995b) to the cis-Golgi network, the medial and trans-Golgi cisternae and the trans-Golgi network (TGN) via vesicular transport. Thus the GA constitutes an obligatory station along the biosynthetic or exo• cytic route for secretory proteins, lysosomal enzymes and integral membrane proteins destined for the plasma membrane or other organelles. Protein sorting and vesicular traffic in the Golgi apparatus 67

Major Routes of Protein and Membrane Traffic To and From the Goigi Apparatus

~"'~ COP II ncaveolln

Figure 1. Diagram depicting the major routes of vesicular traffic along the exocytic (1-10) and endocytic (II-IS) pathways. Exocytic pathways: Secretory proteins, membrane glycoproteins and lysosomalenzymes are synthesized on polyribosomes and are translocated to the ER (I) where they undergo cotranslational and posi-translocational processing (e.g. disulfide bond fonnation, glycosylation, trimming, and oligomerization in some cases). They exit the ER via COP II vesicles (2) that shuttle them to the ER-Golgi intennediate compartment (ERGle). From there they are transported to the cis-Golgi network via COP I-coated vesicles (3). COP I-coated vesicles also function in retrograde transport from the cis-Golgi and ERGIC to the ER (4). Subsequently the proteins either traverse the Golgi cisternae one by one via vesicular carriers (5) or transport occurs by cisternal progression (maturation). Retrograde transport is also assumed to take place between the stacked cisternae (6). Sorting occurs in the trans cisterna or TGN: Lysosomal enzymes bind to M6P receptors, are packaged into clathrin-coated vesicles in the TGN, and are delivered either to early (7) or late (7') endosomes. Membrane and secretory proteins are also sorted in the TGN and delivered by exocytosis along the regulated secretory pathway via secretion granules (8) or along the constitutive pathway (9). In polarized secretory cells there is a separate pathway for delivery of vesicles to the basolater~1 domain (10). Endocytic pathways: Several endocytic pathways have also been charted. The best characterized is receptor-mediated endocytosis via clathrin-coated vesicles budding from either the apical domain of the plasma-membrane (PM) (II) and directed to apical early endosomes (AE) or from the basolateral PM domain (II')and directed to basal early endosomes (BE). Recent evidence indicates that there is also exchange between apical and basal endosomes. Many receptors (LDL, transferrin) recycle back to the plasma membrane from early endosomes (12), whereas many ligands are transported from early to late endosomes (LE) to reach Iysosomes (Ly) (13). Other pathways include uptake in non-clathrin coated vesicles (14) or caveolae (15). 68 M.G. Farquhar and H.-P. Hauri

Models ofGolgi organization

In many cells the Golgi apparatus is a continuous ribbon of regions of compact and noncompact stacked cisternae (Rambourg, 1990), whereas in others it is a collection of dozens of small stacks (e.g. in Caco-2 cells). A number of models have been proposed over the years to explain traffic flow through the GA. The only two that still survive are the "stationary cisternae" model and the "maturation" or "cisternal progression" model. The "maturation" or "cisternal progression" model can be traced to Grasse who suggested (Grasse, 1950) that the Golgi cisternae were formed on the cis face and used up in packaging on the trans face. According to this model the GA is a kind of bottling station in which membrane and contents move in synchrony from one side to the other of the Golgi stack. This model was also inherent in the endomembrane or "membrane flow" concept of Morre and coworkers (Morre et al., 1971, 1979). As information on the GA accumulated, new findings were difficult to reconcile with this model, e.g. the finding that membrane proteins turn over much more slowly than secretory proteins, the existence of membrane recycling and, especially, the cytochemical specialization among Golgi cisternae. The "stationary cisternae" model was introduced in 1981 (Farquhar and Palade, 1981) to ex• plain the compartmentalization and specialization of membrane composition among Golgi cister• nae and within the same cisterna. Its key features are as follows: (1) The GA is organized into a stack of three to eight cisternae that are polarized and have a cis or entry face and a trans or exit face; (2) products enter at the cis face and move vectorially across the stack in the cis to trans direction, traversing the cisternae one by one and becoming progressively post-translationally modified in transit; (3) each cisterna or set of cisternae represents a specific subcompartment of distinctive composition; (4) transport to and through the GA is carried out by vesicular carriers coming to and departing from the dilated rims of the cisternae; (5) all products that follow this route are mixed together in transit until they arrive at the TGN where sorting takes place and secretory proteins, lysosomal enzymes and membrane proteins are packaged into separate popu• lations of vesicles; (6) each vesicle population en route to the GA from the ER or departing from the TGN has a distinctive cytoplasmic coat, as diagrammed in Figure 1; and (7) membranes

Figure 2. Golgi region in gonadotropes (gonadotrophic hormone producing cells) of the rat anterior pituitary illu• strating the classical organization of the GA in glandular cells. Both figures show the stacked Golgi cisternae with dense-cored secretory granules forming on the trans side of the stack. A: Concentration of secretory proteins into a forming secretion granule occurs within the TGN (arrow) which is slightly set off from the stack. Note that this cisterna forms part of the regular stack to the right. B: Another Golgi in which concentration of secretory proteins occurs within the transmost cisterna of the Golgi stack (arrow). Mature. fully condensed secretion granules (sg) and clathrin-coated vesicle (cv) are seen on the trans side of the stack in both figures. A partially condensed secretion granule (cg) is also seen on the trans side in "B". Magnifications: (A) x 18.000; (B) x 29,000. Bar=0.25Ilm. From: Farquhar (1991). Protein sorting and vesicular traffic in the Golgi apparatus 69 70 M.G. Farquhar and H.-P. Hauri

Figure 3. Golgi region from a guinea pig exocrine pancreatic cell incubated for 180 min in the presence of I x 10-5 M carbamylcholine (to stimulate secretion). The micrograph illustrates transitional elements (te) which, in hyperstimulated cells, have numerous elongated protrusions (arrowheads) assumed to be vesicles either detaching from or fusing with transitional elements on their way to or from the cis-Golgi cisterna and/or ERGIC. The micrograph also illustrates the tendency of peripheral Golgi vesicles to form tubular structures (t) which are particularly prominent under these conditions. Concentrated secretory products are present in the transmost-Golgi cisternaerrGN (arrowheads) and associated small granules (arrows). Note that hyperstimulation of acinar cells produces secretory granules that are smaller and more irregular than in unstimulated cells and that some of these granules have c1athrin coats (arrows). Magnification: x 41,000. Bar=0.25 ~m. From: Merisko et al. (1986).

recycle at each station - i.e. retrograde transport occurs between the Golgi and ER, between Golgi subcompartments, between endosomes and the GA, and between the plasma membrane and the GA. Thus the GA not only receives biosynthetic products from the ER, but also it receives considerable recycling vesicular traffic from Golgi subcompatments, the plasmalemma, Iyso• somes and endosomes, and sorts and directs this membrane traffic to its correct destinations. The "stationary cisternae" model gained wide support based on results obtained from in vitro systems (Rothman, 1994; Rothman and Orci, 1992, 1996) and has dominated the field since it was introduced. However, at present the "cisternal progression" model is enjoying a comeback Protein sorting and vesicular traffic in the Golgi apparatus 71 in a modified form. This has come about because several recent developments, such as the failure to detect secretory proteins (apoE and albumin) in Golgi vesicles by immunoelectron microscopy (Dahan et aI., 1994), and the lack of t-SNAREs associated with intra-Golgi transport in the yeast genome, are difficult to reconcile with the "stationary cisternae" model as originally formulated. To explain these data several authors (Schnepf, 1993; Bannykh and Balch, 1997; Glick et aI., 1997, personal communication) have proposed that there is no anterograde vesicular transport. Rather, cisternae move across the stack and gradually mature, and membrane proteins are re• trieved by retrograde vesicular transport, thus maintaining the specialization of Golgi membranes. The current, modified model represents a hybrid between the "cisternal progression" and "vesi• cular transport" models as only anterograde vesicular transport between Golgi cisternae (route 5 in Fig. 1) is eliminated whereas anterograde transport between ER and GA, between the Golgi and plasma membrane, and retrograde transport between Golgi cisternae and between GA and ER is assumed to be vesicular. The major evidence cited in support of the "cisternal progression" model is the biogenesis of algal scales originally described in the 1970s (Brown, 1971; reviewed recently by Becker et aI., 1995) which takes place within a single cisterna and, more recently, the assembly of procollagen (Leblond, 1989) and casein (Clermont et aI., 1993). It is safe to say that neither the "stationary cisternae" nor the "cisternal progression" model for anterograde trans• port is completely proven.

Mechanisms of sorting, targeting and transport from the ER

Exitfrom the ER occurs at specific export sites

It was recognized some time ago (Palade, 1975) that exit of newly synthesized proteins from the ER (I) occurs at specific sites in cells specialized for secretion, - i.e. the part rough-part smooth transitional elements of the ER which have characteristic protrusions assumed to represent bud• ding vesicles (Fig. 3), and (2) takes place via small vesicles or tubules typically concentrated in clusters located between the ER exit sites and the cis side of the GA. In other cell types, not specialized for secretion, similar specialized regions of the ER (Fig. 4) associated with vesicular protrusions and clusters of vesicles and tubules (called vesicular tubular clusters or VTCs) are also recognized (Balch et aI., 1994; Stinchcombe et aI., 1995; Bannykh et aI., 1996; Lotti et aI., 1996), but transitional elements are not so highly developed as in secretory cells with a prominent regulated secretory pathway. It has also become apparent that exit can occur both in close proxi• mity to the GA and also at multiple sites more peripherally located in the cell (Saraste and Svensson, 1991; Lotti et al., 1996; see Fig. 6). 72 M.G. Farquhar and H.-P. Hauri

The most widely-accepted working model at present is that 1) these tubulovesicular clusters located between the ER and cis-Golgi network correspond to ERGIC and include molecules delivered from the ER via COP II-coated vesicles, and 2) from ERGIC cargo is then transported either to the cis-Golgi or recycled back to the ER in COP I-coated vesicles (Pind et al., 1994; Stinchcombe et al., 1995; Bannykh et al., 1996).

Selective transportfrom the ER vs bulk-flow models

In 1987 Rothman and colleagues (Pfeffer and Rothman, 1987; Wieland et al., 1987) proposed the "bulk-flow theory" which holds that transport out of the ER is nonselective, and that sorting occurs through retrieval mechanisms. This theory became the dogma for some time but recently has been challenged. There is now growing evidence that exit from the ER is selective - i.e. that mature, properly-folded secretory (Mizuno and Singer, 1993) and membrane (Balch et aI., 1994; Aridor and Balch, 1996; Bannykh et aI., 1996) proteins are sorted for packaging into carrier ve• sicles rather than being transported by bulk-flow. Evidence was obtained by quantitative immuno• electron microscopy that VSV-G protein is concentrated nearly tenfold during export from the ER in vitro (Balch et aI., 1994; Bannykh et aI., 1996). Moreover, it is now recognized by the authors (Rothman and Wieland, 1996) that the original data for the bulk-flow hypothesis was based on inappropriate assumptions regarding the rate of movement of putative peptide bulk-flow markers relative to secreted proteins. These data have now been re-evaluated and found to favor selective transport (Rothman and Wieland, 1996). The question that arises is, how is sorting and concentration of proteins for export from the ER accomplished? First, according to prevailing working models, resident ER protein are retained in the ER and excluded from the vesicles by ER retention signals (Pelham, 1989). Second, it is envi• saged that sorting is accomplished by receptors or sorting chaperones that bind the transported proteins and at the same time bind cytosolic coat complexes (Aridor and Balch, 1996; Bednarek et aI., 1996). The model for this scenario is clathrin coat assembly where endocytic receptors interact with adaptins driving the formation of clathrin-coated pits. A candidate sorting receptor for at least some proteins is the transmembrane protein, Emp24, a component of ER-derived

Figure 4. Putative transitional elements and transport intermediates between the rough ER and Golgi complex in HEp-2 cells transfected with HRP cDNA carrying a KDEL retrieval signal (HRP KDEL) and incubated at 20 DC (A) or at 20 DC and shifted to 37 DC (B, C). HRP is seen within the putative transitional elements (te) as well as the cluster of vesicles (VTC) which includes anterograde as well as retrograde transporting vesicles. An increase in tubular profiles is seen in cells incubated at 20 DC (arrows in "A"). Magnifications: (A) x 15,500; Bar=0.25 Jlm; (B) x 38,000; Bar=O.1 Jlm; (C) x 38,000; Bar=O.l Jlm. From: Stinchcombe et al. (1995). Protein sorting and vesicular traffic in the Golgi apparatus 73 74 M.G. Farquhar and H.-P. Hauri vesicles in yeast which is required for selective transport of a subset of proteins from the ER and thus has the expected characteristics of a cargo receptor (see Chapter by Duden and Schekman, this volume). This protein belongs to a family of proteins with homologues in yeast and humans (Wade et al., 1991; Starnnes et al., 1995).

Transit through the ER-Golgi intermediate compartment (ERGlC)

There is now widespread agreement (Pelham, 1991; Hauri and Schweizer, 1992) that, in mam• malian cells, newly synthesized proteins pass through ERGIC, also referred to as the "salvage compartment", "vesicular tubular clusters" or "VTCs" (Balch et aI., 1994) (Fig. 5), 15° com• partment (Saraste and Kuismanen, 1992), and, most recently, "sorting exosomes" (Aridor and Balch, 1996), interposed between the transitional elements of the ER and the stacked Golgi cister• nae en route to the cis-Golgi network. This compartment differs in composition from that of either ER or Golgi membranes and is marked by the presence of p53, now called ERGIC-53 (Figs 6 and 7), (Schweizer et aI., 1988; Schindler et aI., 1993) or its rat homologue, p58 (Saraste et aI., 1987; Lahtinen et aI., 1996). Opinions vary as to the precise morphology of this compart• ment and whether one or two steps of vesicle formation and fusion are required for transit through it. Recent studies in vitro in mammalian cells have provided evidence that two steps are required - the first carried out by COP II-coated vesicles which are solely responsible for export from the ER, and the second by COP I-coated vesicles. COP I is recruited onto vesicles of the intermediate compartment where it initiates segregation of anterograde transport (VSV-G protein) from retrograde transported protein (p58) (Pepperkok et aI., 1993; Aridor et aI., 1995). In yeast both COP I and COP II-coated vesicles have been shown to bud from the perinuclear cisternae in vitro (see the chapter by Duden and Scheckman, this volume). However, in mammalian cells no evidence has been found for involvement of COP I-coated vesicles in transport out of the ER (Aridor and Balch, 1996).

Figure 5. Transport of VSV-G protein from the ER to VTCs (ERGlC) and to the Golgi stack in digitonin-permea• bilized NRK cells. NRK cells were infected with mutant VSV (ts045) at the non-permissive temperature (39°C) for 2 h, permeabilized with digitonin, shifted to the permissive temperature (32°C), and incubated in the presence of an ATP-generating system and cytosol for varying periods. Prior to incubation of digitonin-permeabilized cells in vitro, VSV-G is confined to the ER cisternae (arrowheads) and nuclear envelope (arrows in "A"). After incubation for IO min in vitro at 32°C (B, C), VSV-G is found in small vesicles, and at 15 min (D-F) it is found in VTCs which correspond to ERGlC, but not in the GA. By 30 min incubation in vitro (G) it has reached the cis face of the GA. By 60 min (H) VSV-G is found on both the cis and trans side of the stack and throughout the stack. In the presence of a mutant rabla (Rabla (NI241)) incapable of binding either GDP or GTP (E), EDTA (F), or GTP)'S (not shown), VSV-G fails to be transported to the Golgi stack and accumulates in ERGIC (VTCs). Magnifications: (A) x 40,000; Bar=O.1 ~m; (B-C) x 150,000; Bar=0.02 ~m; (D-F) x 80,000; Bar=0.05 ~m; (G-H) x 100,000; Bar=0.05 ~m. A-C and H are from Balch et al. (1994). E and G are from Pind et al. (1994). Protein sorting and vesicular traffic in the Golgi apparatus 75 76 M.G. Farquhar and H.-P. Hauri

Other proximal pre-Golgi, pre-ERGIC, smooth ER compartments

In addition to the ERGIC a number of specialized smooth membrane structures have been de• fined that are typically located proximal to the intermediate compartment and are in continuity with the rough ER (see Hauri and Schweizer, 1997, for a review). Some of these typical smooth ER compartments occur naturally, such as the tubular smooth ER of the hepatocyte which is the site of concentration of enzymes involved in detoxification (cytochromes P450 and b5, epoxide hydrolase) (Galteau et aI., 1985; Marti et aI., 1990), the smooth ER of steroid-secreting cells assumed to be the site of concentration of ER enzymes involved in cholesterol synthesis (Andreis et aI., 1989; Mendis-Handagama et aI., 1989), the smooth ER in muscle cells associated with se• gregation and amplification of calcium-binding proteins (Sitia and Meldolesi, 1992), the smooth• surfaced stacks found in Purkinje neurons believed to be sites of high levels of expression of the inositol trisphosphate receptor (Satoh et aI., 1990; Villa et aI., 1991), and a system of smooth tubules found in chondrocytes believed to be the site of concentration and assembly of proteo• glycan precursors (Vertel et aI., 1989). In addition to these naturally occurring regions of smooth ER, a number of experimentally• induced smooth membrane compartments have been described, such as the "crystalloid smooth ER" found in mutant CHO cells synthesizing massive amounts of the resident ER membrane protein, HMG CoA reductase (Chin et aI., 1982), flattened smooth cisternae seen in COS cells expressing a rat growth hormone-influenza hemagglutinin chimeric protein (Rizzolo et aI., 1985), and the extensive networks of smooth membranes found in cells overexpressing rubella virus El glycoprotein (Fig. 8) (Hobman et aI., 1992) and in thymic epithelial cells from mice with a null mutation for the MHC-encoded peptide transport, TAP (Raposo et aI., 1995). The latter three examples are of particular interest in the present context because in these cases the biosynthetic products are either improperly folded (growth hormone chimera or HLA-B27 molecules) or represent unassembled monomers (rubella El glycoprotein) and are unable to exit the ER. The amplified smooth membranes have properties different from those of the ER or ERGIC and are believed to represent populations of smooth membranes located at or near the exit site of the ER which become amplified under these conditions (Hobman et aI., 1992). It appears that the ex-

Figure 6. Visualization of the ERGIC and the Golgi apparatus by confocal laser scanning, double immunofluores• cence microscopy in normal human fibroblasts (MRC-5). ERGIC was labelled with a monoclonal antibody against ERGlC-53 (C) (Schweizer et a\., 1988) and the GA with a rabbit polyclonal antibody against galactosyl• transferase (B) (Roth and Berger, 1982). Superimposition of the two images (A) shows ERGIC elements both near the Golgi stack and at the cell periphery. Cells were fixed in paraformaldehyde, permeabilized with Triton X-IO and processed for indirect fluorescence microscopy using F1TC- and rhodamine-labeled secondary antibodies. Magnification: (A) x 400; (C) x 200; Bars: 10 Ilm. (Micrographs provided by Dr. Thomas Bachi, University of ZUrich, ZUrich, Switzerland). Protein sorting and vesicular traffic in the Golgi apparatus 77 78 M.G. Farquhar and H.-P. Hauri

Figure 7. Localization of ERGIC-53 protein by immunogold electron microscopy on an ultrathin cryosection of a human intestinal enterocyte. ERGIC-53 is predominantly localized in tubulovesicular clusters (boxes) constituting the ER-Golgi intermediate compartment near the cis-Golgi. G = Golgi stack; n= nucleus. Magnification: x 20,000; Bar=0.5 ~m. From: Schweizer et al. (1988). Protein sorting and vesicular traffic in the Golgi apparatus 79 pressed proteins travel to the distal extensions of the ER where they accumulate and are finally degraded. In the case of the thymic epithelial cells of TAP-deficient mice, this smooth ER com• partment represents the site where misfolded proteins are eventually degraded (Raposo et al., 1995). This and other recent work (Bonifacino, 1996; Wiertz et aI., 1996) has demonstrated that transport-incompetent ER proteins destined for degradation are translocated across the ER membrane to the cytosol by the Sec61-containing translocon in a process known as reverse trans• location. Once exposed to the cytosol they are deglycosylated, released from the ER membrane, polyubiquinated and delivered to proteosomes for degradation. These findings raise the possibi• lity that the amplified smooth ER compartments seen under circumstances in which transport• incompetent proteins are overexpressed, may represent both the ER exit site and the site of reverse translocation.

Mechanisms for retention and retrieval ofresident ER proteins: Current models

A key question is, how are resident ER proteins retained in the ER while itinerant proteins are allowed to move down the secretory pathway to pre-Golgi and Golgi compartments? According to the prevailing point of view both membrane proteins and soluble proteins of the ER remain localized in this organelle by a combination of retention and retrieval mediated through ER tar• geting signals (Pelham, 1989). The best characterized ER targeting signal is the C-terminal KDEL or closely related sequence (HDEL, RDEL or KEEL, etc.) found on many soluble resi• dent ER proteins such as BiP and protein disulfide isomerase. This C-terminal sequence is neces• sary and sufficient for ER retention because removal of the KDEL signal from BiP caused it to be secreted (Munro and Pelham, 1986), and addition of this sequence was sufficient to retain lysozyme, a secreted protein, and cathepsin D, a lysosomal enzyme, in the lumen of the ER (Pelham, 1988). How does the KDEL motif serve as a localization signal? According to the current prevailing opinion the KDEL sequence serves as a retrieval signal for soluble ER resident proteins that escape the ER (Sonnichsen et aI., 1994). When luminal ER proteins escape the ER they bind to a specific KDEL receptor, a 23 kD membrane protein with six or seven transmembrane domains (Tang et aI., 1993; Scheel and Pelham, 1996), which is concentrated in transport vesicles, ERGIC and the cis-Golgi cisterna (Griffiths et aI., 1994). In this way soluble ER proteins are prevented from passing down the exocytic pathway and are returned to the ER via COP I-coated retrograde transport vesicles recycling from the GA to the ER (Pelham, 1995). Since the number of recep• tors is only about one tenth that of ER proteins with KDEL sequences the retrieval mechanism is considered to be a fail-safe mechanism, and most ER proteins are thought to be retained in the ER 80 M.G. Farquhar and H.-P. Hauri by binding to an ER matrix (Sonnichsen et aI., 1994) composed of a network of chaperones (Wada et aI., 1991; Tatu and Helenius, 1997), many of which are calcium-binding proteins (Sambrook, 1990; Sonnichsen et aI., 1994). It has been proposed that Ca2+ may regulate the assembly and disassembly of the complexes (Sambrook, 1990; Wada et al., 1991). A similar scenario appears to operate for retention and retrieval of at least some ER membrane proteins which possess either a C-terminal, di-Iysine (KKXX) motif found on type I membrane proteins (Jackson et al., 1990) or a di-arginine (XXRR) motif in the case of type II membrane proteins (Schutze et aI., 1994). These signals function to retrieve ER membrane proteins from the GA (Jackson et aI., 1993; Teasdale and Jackson, 1996). Analysis of yeast mutants has shown that the di-Iysine signal binds directly to coat proteins of COP I-coated vesicles, suggesting that the coat collects the proteins into COP I-coated vesicles much in the same way as clathrin-containing coats concentrate endocytic receptors into coated pits (Cosson and Letourneur, 1994; Letourneur et aI., 1994). These studies provided the first evidence that COP I-vesicles function in retrograde, Golgi to ER transport.

Transport, processing and sorting by the GA

After leaving the ERGIC, COP-coated vesicles with their cargo of newly-synthesized proteins fuse with the first cisterna on the cis side of the Golgi stack, often referred to as the cis-Golgi network. According to the current working model (see Fig. 1), the proteins are then transported via vesicular transport successively to each cisternae or set of cisternae in the Golgi stack, which are presumed to represent compartments of distinctive composition, and they are extensively post• translationally modified in transit by Golgi-specific enzymes as they move through each compart• ment. The evidence available suggests that transport through the Golgi system is by nonselective "bulk-flow" mechanisms and does not involve sorting and concentration (Griffiths et aI., 1984; Quinn et aI., 1984; Balch et aI., 1994). Thus cargo proteins are mixed together in the transport vesicles and stacked cisternae until reaching the TGN where sorting takes place.

Figure 8. Novel post-rough ER, pre-ERGIC compartment where unassembled monomers ofrubella virus EI glyco• protein accumulate in CHO cells stably expressing EI. (A) This compartment consists of a network of tubular smooth membranes (tn). (B) There are points of continuity between rough ER cisternae (rer) and the tubular smooth network (arrowheads) located in close proximity to Golgi cisternae (Gc). (C) Immunogold localization of EI glycoprotein in the tubular network of smooth membranes. Magnifications: (A) x 15,000; Bar=0.5 Jlm; (B) x 24,000; Bar=0.5 Jlm; (C) x 20,000; Bar=0.25 Jlm. From: Hobman et al. (1992). Protein sorting and vesicular traffic in the Golgi apparatus 81

-rer / 82 M.G. Farquhar and H.-P. Hauri

Contributions ofin vitro assays

Progress in defining the mechanisms of vesicular transport to and through the GA has been quite rapid during the last ten years due largely to the introduction of in vitro systems (both cell-free systems and permeabilized cells) in which the molecular requirements involved could be defined, purified, and their effect on transport assessed. Assays are now available for studies of ER to Golgi transport (Beckers et aI., 1987) (see Fig. 5), intra-Golgi transport (Rothman and Orci, 1992), and for budding from the TGN (Simons and Virta, 1987; Salamero et al., 1990; Tooze and Huttner, 1990; Xu and Shields, 1993; Simon et aI., 1995; Wang et aI., 1995). All these assays depend on the presence of cytosol and ATP-generating systems, and the cytosol can be frac• tionated for identification of required components. For example, several of the proteins that are key players in intra-Golgi transport, such as NSF (N-methylma1eimide sensitive factor), an ATPase that stimulates transport, a and y SNAP (soluble NSF attachment protein), required for binding NSF to membranes, and coat components (see below), were defined by fractionation of the cytosol, purification of the components critical for transport and assessment of their activity in in vitro assays (Rothman and Orci, 1992; Rothman, 1994). Also, not only the depleted proteins but other reagents such as antibodies, signalling molecules and various inhibitors can be added back to the assay to assess their effect on transport. In vitro assays have been particularly power• ful when coupled with morphological analysis and immunocytochemistry to assess the stage of vesicle budding, docking and fusion and to localize precisely in the vesicle, donor membrane, or target membrane, the proteins involved (Rothman and Orci, 1992; Balch et aI., 1994; Pind et al., 1994; Rothman and Orci, 1996; Schekman and Orci, 1996).

The SNARE hypothesis

In 1993 progress in understanding the molecular requirements for vesicular transport was rapidly accelerated by the convergence of information derived from the in vitro studies mentioned above, analysis of yeast mutants (Pryer et aI., 1992), and characterization of components of synaptic vesicles (Bennett and Scheller, 1993; Bennett and Scheller, 1994; Stidhof, 1995). Analysis of yeast mutants had revealed that many of the proteins that were essential for different steps in vesicular transport in yeast were homologues of synaptic vesicle proteins or of the fusion proteins NSF (Sec 18P) and aSNAP (Sec 17P). Rothman and colleagues (Sollner et aI., 1993a) showed that recombinant NSF, aSNAP, and ySNAP formed a 20S complex with the synaptic vesicle• associated proteins syntaxins, SNAP-25 (no relationship to a- and y-SNAP), and VAMP/ syna• tobrevin 2 when mixed with a detergent-solublized membrane extract from bovine brain. Based Protein sorting and vesicular traffic in the Golgi apparatus 83 on these and earlier experiments these investigators (Sollner et aI., 1993a; Sollner et aI., 1993b) put forth the SNARE hypothesis, which proposes that every transport vesicle contains on its surface a specific protein or v-SNARE that binds to a compartment-specific t-SNARE on its target membrane. Since the introduction of the SNARE hypothesis a number of such putative v-and t-SNARES have been identified as homologues of syntaxin, SNAP-25 and VAMP and shown to be required for specific vesicular transport steps such as ER to GA, GA to vacuole and GA to plasma mem• brane, particularly in yeast (see Chapter by Duden and Scheckman, this volume) but also in mammalian cells (see below). Recent evidence suggests that the original SNARE hypothesis will probably have to be refined, modified and expanded (see below).

Golgi compartments and post-translational processing oftransported proteins

Golgi-specificfunctions The list of post-translational modifications that are known to occur exclusively in Golgi subcom• partments includes terminal N-glycosylation of glycoproteins (trimming of mannoses and addi• tion of N-acetyl glucosamine, galactose, fucose, and sialic acid) and glycolipids (Kornfeld and Kornfeld, 1985); O-glycosylation of glycoproteins; addition of the mannose 6-phosphate recog• nition marker to lysosomal enzymes; and sulfation of proteoglycans, oligosaccharides, and tyro• sine. In addition, sphingolipids are synthesized in the GA (reviewed by Allan and Kallen, 1993). The TGN and transmost cisterna are the site of concentration of secretory proteins, and proteo• lytic processing of prohormones can begin in the TGN (Schnabel et aI., 1989; Lepage-Lezin et aI., 1991; lung et aI., 1993). Many of the enzymes involved in these functions have been used as general Golgi markers and to define Golgi subcompartments for both biochemical and immuno• cytochemical studies (see Figs. 6B and 8). Analysis of the distribution of enzymes responsible for trimming and addition of sugars to N-linked oligosaccharides has played a key role in understanding the topography of the GA: it gave the first clues that Golgi enzymes are laid out in space sequentially as they act over time (see Farquhar, 1985 for a review; see also chapter by Roth, this volume).

How many Golgi compartments are there? At present we define four Golgi compartments - cis (or CGN), medial, trans and TGN (see Fig. 1) based on the distribution of marker enzymes. Information available on the location within Golgi compartments of the major Golgi-specific membrane proteins, about which there is reason• able agreement, is given in Table I. 84 M.G. Farquhar and H.-P. Hauri

Table I. Localization of membrane proteins to Golgi compartments in mammalian cells

Goigi Compartment Protein FunctionlFeatures References cis (CGN) Peptide-GaINAc- Initiation of protein (Roth et aI., 1994) (i.e. first cis-Golgi transferase O-glycosylation cisterna) GlcNAc Phosphotrans- Initiation of mannose 6-phos- (Kornfeld and Kornfeld, 1985; ferase* phate signal attachment to Iy- Schweizer et al., 1991) sosomal enzymes KDEL-receptor In part localized to the ERGIC (Tang et aI., 1993; Griffiths at steady state; recycles to ER et aI., 1994) upon binding to KDEL-proteins medial a-mannosidase It First Goigi enzyme involved in (Velasco et aI., 1993) complex glycosylation; often considered a cis-Goigi marker GIcNAc-transferase I Involved in complex glycosy- (Dunphy et aI., 1985; Nilsson lation et aI., 1993a) a-mannosidase nt Involved in complex glycosy- (Velasco et aI., 1993) lation Phosphodiesterase* Second enzyme involved in (Kornfeld and Kornfeld, 1985; mannose 6-phosphate signal at- Schweizer et aI., 1991) tachment to lysosomal enzymes Giantin C-terminally anchored, largest (Lindstedt and Hauri, 1993; Goigi protein; also present in Seelig et aI., 1994) cis-Goigi GPPI30/GIMPc Calcium-binding (Yuan et aI., 1987; Lindstedt phosphoprotein; retained in the and Hauri, 1997) GA by a pH-dependent mechanism MG-160 May recycle between medial- (Gonatas et aI., 1989) and trans-Goigi trans ~-1,4- Involved in complex glycosy- (Roth and Berger, 1982; Nilsson galactosyltransferase lation et aI., 1993a) TGN a-2,6-sialyltransferase Involved in complex glycosy- (Roth et aI., 1985; Rabouille lation; also present in trans- et aI., 1995) Goigi TGN-38 May be involved in vesicle for- (Luzio et aI., 1990) mation at the TGN; recycles between TGN, plasma membrane and endosomes Furin Endoprotease that cleaves pre- (Bosshard et aI., 1994) cursor proteins at dibasic sites CI- and CD-mannose Only partially localized in the (Geuze et aI., 1984; Griffiths 6-phosphate receptors TGN; major localization is late et aI., 1988; Woods et aI., 1989) endosome Tyrosylprotein/sulfo- Sulfate attachment to tyrosine; (Huttner, 1988; Niehrs and transferase* is the last protein modification Huttner, 1990) in the Goigi in most cells

* These proteins have not been localized yet by irnmunoelectron microscopy. t Localization is somewhat variable in different cell types: medial only in NRK and CHO cells, medial plus trans in pancreatic acinar cells and enterocytes, or across the entire stack in hepatocytes and some enterocytes. Protein sorting and vesicular traffic in the Golgi apparatus 85

Some time ago three Golgi subcompartments - cis, medial and trans - of distinctive composi• tion were defined, based on the ability to separate lysosomal GlcNAc phosphotransferase, ct.• mannosidase IT, and galactosyltransferase on analytical sucrose density gradients (Dunphy and Rothman, 1983; Goldberg and Kornfeld, 1983). Later, based on morphological and immunocyto• chemical studies, the TGN was defined as a separate subcompartment - distinct from the trans• Golgi cisternae - responsible for packaging and sorting of newly synthesized proteins into different carrier vesicles. This increased the total to four (Griffiths and Simons, 1986). Some• times a fifth subcompartment, the cis-Golgi network, has been defined, but our view is that the cismost Golgi cisterna and the cis-Golgi network describe the same first or entry compartment of the GA. In the past there has been variation in opinions concerning the identification of the cis• Golgi network and the intermediate compartment (Hauri and Schweizer, 1992; Mellman and Simons, 1992). Less information is available on the localization of enzymes involved in O-glycosylation but the current consensus is that addition of N-acetyl galactosamine, the first step in O-glycosylation, occurs in the cis-Golgi compartment (Roth et al., 1994; Schweizer et aI., 1994). Localization of glycosylation enzymes will not be discussed further here as they are reviewed in detail in Chapter 5. Sulfation is a late Golgi event associated with the TGN or with secretion granules (Huttner, 1988). Addition of the mannose 6-phosphate (M6P) recognition marker to lysosomal enzymes is an early (cis-Golgi) event, and it occurs in two steps (Kornfeld and Mellman, 1989). First, a phos• photransferase transfers N-acetylglucosamine I-phosphate from UDP-GlcNAc to one or more mannose residues on lysosomal enzymes to give rise to a phosphodiester intermediate. Then a phosphodiester glycosidase removes the GlcNAc residue to expose the Man6P sorting signal. Biochemical evidence suggests that both the phosphotransferase and phosphodiester glycosidase activities are associated with a post-ERGIC compartment, probably the cis-Golgi (Schweizer et aI., 1991). How do the enzymes that add the M6P signal recognize lysosomal enzymes and distinguish them from other N-linked glycoproteins? There is evidence that this is accomplished by the presence of a recognition marker formed by a patch of lysine residues present on lyso• somal enzymes but not on other proteins (Kornfeld and Mellman, 1989) in cooperation with other regions of lysosomal enzymes (Dustin et aI., 1995). Concepts concerning the existence of Golgi subcompartments were initially rather rigid and each Golgi stack was believed to have an identical arrangement. However, morphological studies documented considerable differences in the organization of the GA among different cell types (Farquhar and Palade, 1981) (see Figs 2 and 3) and that Golgi organization can change drama• tically upon experimental manipulation (e.g. low temperature, ATP depletion, viral infection; re• viewed in Farquhar, 1991). More recently, immunocytochemical studies (Roth et aI., 1986; 86 M.G. Farquhar and H.-P. Hauri

Nilsson et aI., 1993a; Velasco et aI., 1993; Rabouille et aI., 1995) as well as biochemical data on Golgi fractions (Hayes et aI., 1993; Hayes and Varki, 1993; Sjoberg and Varki, 1993) have docu• mented that there is considerable overlap in enzyme activities between one or more Golgi sub• compartments as well as differences in the distribution of Golgi processing enzymes from one cell type to another (see Fig. 9).

Figure 9. Localization of Man II in the pancreatic acinar cell by immunogold labeling of ultrathin cryosections. (A) Gold particles are seen across the Golgi stack (Gc) but are most numerous in medial- and trans-cisternae and a condensing vacuole (cv). (B) A similar distribution of label is seen with antibody affinity-purified on deglycosylated Man II. er: endoplasmic reticulum; m: mitochondria; zg: zymogen granule. Magnifications: (A, B) x 28,000; Bar=0.25 11m. From: Velasco et al. (1993).

Proposed mechanismsfor retention and retrieval ofresident Golgi proteins

The existence of at least four Golgi compartments with different functions suggests that each compartment possesses a distinct complement of resident proteins. The question is, how are these resident proteins directed to the correct region of the GA and maintained there? In the case of the Protein sorting and vesicular traffic in the Golgi apparatus 87

TGN there is evidence for a retrieval mechanism comparable to that for ER membrane proteins: TGN-resident proteins, such as TGN38, furin and yeast proteases (Kex2p, Kexlp, and dipeptidylaminopeptidase A), cycle between the plasma membrane and the TGN and are retrieved by a tyrosine localization signal in their cytoplasmic tails (Humphrey et aI., 1993; Nilsson and Warren, 1994; Marks et aI., 1997) similar to those found in endocytic receptors (see Table 2). For example, transplantation of the tyrosine-based signal of TGN38, YQRL, to reporter mole• cules results in their localization to the TGN.

Table 2. Consensus sequence motifs for targeting to clathrin-coated vesicles

Motif* Protein Destination

NPXY-type: FDNPV y LDL receptor Early endosome SVNPEY IGP-I receptor Early endosome FEN TLY Mannose receptor Phagosome YEN PTY ~-amyloid precursor protein ? GENP J: Y ~-I integrin ?

YXX0-type: YTRF Transferrin receptor Early endosome Y Q DL Asialoglycoprotein receptor HI Early endosome YSKV CI Mannose 6-phosphate receptor Late endosome YRGV CD Mannose 6-phosphate receptor Late endosome GY Q T J: Lampl Lysosome GYE Q F Lamp 2 Lysosome GYEVM CD 63 Lysosome GYRHV Acid phosphatase Lysosome Y Q PL T-cell receptor (CD3) Lysosome YTPL HLA-DM-beta MHC class II compartment YQN J: CDlb MHC class II compartment Y Q RL TGN38 TGN YSAF Poly-immunoglobulin receptor Basolateral plasma membrane YSPL HNgpl60 ?

Di-Ieucine-type: LL Pc receptor Lysosome L J: MHC class II invariant chain MHC class II compartment

K-KIR-FIY-FlY-type: KKFF ERGIC-53t Lysosome KRFY VIP36 TGN and post-Golgi vesicles

* Bold type amino acids indicate the key features defining the motif. The intermediate compartment marker protein ERGIC-53 is in part transported to the cell surface at very high expression levels of this protein in transfected cells and then internalized by clathrin-mediated endocytosis to be degraded in lysosomes (Kappler, Itin, Hopkins, and Hauri, unpublished). o Bulky hydrophobic amino acid. 88 M.G. Farquhar and H.-P. Hauri

The bulk of the evidence available suggests that localization of proteins in the Golgi stack is accomplished by retention rather than retrieval and involves a combination of both protein-protein interactions (through the transmembrane as well as the luminal or cytoplasmic domains) and protein-lipid interactions. No single mechanism or signal appears to be at work (Machamer, 1996). Several different mechanisms and models have been proposed to explain how resident proteins of the Golgi stacks are retained - i.e. transmembrane domain signals, formation of large insoluble aggregates, kin recognition, and bilayer-mediated sorting.

Transmembrane domain retention signals That targeting information resides in the transmembrane domain was originally documented for the coronavirus (mV) M protein (formerly called El) (Machamer, 1993), a glycoprotein with three transmembrane domains, in which only the first transmembrane domain is needed for reten• tion in the cis-Golgi (Machamer and Rose, 1987; Machamer et aI., 1990). Since then the same has been demonstrated for a number of endogenous Golgi enzymes such as ~-1 ,4-galactosyl• transferase (~-1 ,4-GT), a-2,6-sialyltransferase (a-2,6-ST), and ~-acetylglucosaminyltransferase I (GlcNAc transferase I), all of which are type II membrane proteins (Colley, 1997). There is no primary sequence homology in the transmembrane domains of any of these proteins. Transplan• tation of the transmembrane domain of ~-1 ,4-GT was sufficient to confer localization in the trans-Golgi cisternae to ovalbumin (Teasdale et aI., 1992) and the invariant chain (Nilsson et aI., 1991). Similarly, the transmembrane domain of GlcNAc transferase I was sufficient for localizati• on of dipeptidylpeptidase IV, a plasma membrane protein, to the medial-Golgi (Burke et al., 1992; Tang et aI., 1992) and a-2,6-ST to the trans-Golgi (Wong et aI., 1992). There seems to be gener• al agreement in the case of these and other Golgi proteins that information important for targeting resides in the transmembrane domains. However, in some cases hydrophilic sequences flanking the transmembrane domain are also required for Golgi localization (Munro, 1991).

Formation ofinsoluble aggregates too large to enter transport vesicles This model was based on the finding that a chimeric protein consisting of the ectodomain of VSV-G protein and the first transmembrane domain of the coronavirus M protein (which is re• tained in the cis-Golgi) forms large, detergent-resistant oligomers (Weisz et aI., 1993). However, this idea has lost favor because there is no evidence that large oligomers of Golgi membrane proteins exist within the lipid bilayer, and it has been shown that the active forms of many of the medial- and trans-Golgi enzymes are dimers (a-2,6-ST; ~-I,4-GT, UDPase) or monomers (N• deacetylaselN-sulfotransferase) (Mandon et aI., 1994; Machamer, 1996). The possibility exists that aggregation of some Golgi membrane proteins occurs as a result of altering the lipid environ• ment by detergent solubilization. Protein sorting and vesicular traffic in the Golgi apparatus 89

The kin recognition model This model is also based on the concept that resident proteins of a particular cisterna interact to form large hetero-oligomers that restrict forward movement (Nilsson et a!., 1993b; Nilsson and Warren, 1994). It proposes that Golgi enzymes, many of which are homodimers with their lumi• nal domains bound together, are retained in the GA via protein-protein interactions between the hydrophobic transmembrane domains of adjacent "kin" dimers - i.e. other resident proteins of the same compartment. As a result large linear hetero-oligomers form which become attached to an underlying cytoskeletal, intercisternal matrix (Nilsson et a!., 1993b). This model, originally proposed for medial-Golgi enzymes, was based on the finding that when the medial-Golgi enzyme GlcNAc transferase I was held in the ER by the addition of an ER-specific retention signal, another medial-Golgi enzyme, a-mannosidase II, also accumulated in the ER in large aggregates whereas enzymes of the trans cisternae (e.g. a-2,6-ST), did not accumulate. This model is difficult to rationalize with recent observations on the behavior of Golgi proteins tagged with green fluorescent protein in living cells demonstrating that Golgi resident proteins are highly mobile and diffuse rapidly and freely in Golgi membranes (Cole et a!., 1996). Moreover, this model does not seem to apply to trans-Golgi enzymes, as interaction between two trans-Golgi enzymes a-2,6-ST and p-l A-GT, was not detected in a similar assay (Munro, 1995). Also, even in the case of a-mannosidase II and GlcNAc transferase I, interaction occurs through their luminal domains (Munro, 1995; Nilsson et a!., 1996).

Bilayer-mediated sorting This model proposes that the length of the transmembrane domain is the key factor in sorting of resident Golgi proteins (Bretscher and Munro, 1993; Munro, 1995) and is based on the observa• tion that endogenous Golgi enzymes have shorter (17 residues on average) transmembrane domains than plasma membrane proteins (21 to 22 residues). According to this model the two sets of transmembrane domains confer different behaviors by virtue of differences in their physi• cal properties. It proposes that there are mixed lipid populations within the bilayer of a Golgi cisterna that separate into lipid microdomains with distinct composition. The Golgi membrane proteins with shorter transmembrane regions are retained because they partition into the thinner cholesterol/sphingolipid-poor microdomains and are excluded from cholesterol-rich regions of Golgi membranes en route to the plasma membrane. Support for this model was provided by the findings that lengthening of the transmembrane domain by five or more residues greatly reduced retention, and reducing the length of the transmembrane domain of a plasma membrane protein by six residues resulted in its localization to the GA (Munro, 1995). To summarize, several different localization mechanisms have been proposed for retention of membrane proteins in the Golgi stack. None of the mechanisms or models proposed is sufficient 90 M.G. Farquhar and H.-P. Hauri to explain the targeting of all Golgi proteins, and none is mutually exclusive. What appears to be agreed upon and is so far unique is the importance of the transmembrane domain in Golgi retention.

Sorting at the TGN

Currently it is believed that all newly synthesized proteins and glycolipids pass through the entire Golgi system until they reach the final compartments of the GA - the trans-cisternae and TGN • where they are sorted and packaged into different populations of vesicles destined for different locations (Griffiths and Simons, 1986; Burgess and Kelly, 1987). The TGN is the main sorting station in the exocytic pathway. In secretory cells of endocrine and exocrine glands and in neurons, often referred to as "regulated" secretory cells, it has been recognized for some time that three major populations of vesicles bud from the TGN: dense-cored secretory granules whose contents are discharged by regulated exocytosis upon demand (Figs 2, 3 and 10), consti• tutive secretory vesicles carrying secretory proteins and membrane proteins that are continuously delivered to the plasma membrane (Fig. 10), and clathrin-coated vesicles carrying lysosomal enzymes bound to mannose 6-phosphate receptors (MPRs) destined for delivery to Iysosomes via early or late endosomes. In nonglandular, nonpolarized cells such as fibroblasts, often referred to as constitutive secretory cells, only two TGN-derived vesicle populations have traditionally been recognized - one carrying lysosomal enzymes destined for Iysosomes and another assumed to carry both constitutively secreted membrane and secretory proteins (Burgess and Kelly, 1987). However, recently evidence has been obtained suggesting that a cryptic, calcium-regulated secre• tory pathway is also present in "constitutive" secretory cells such as fibroblasts (Steinhardt et aI., 1994; Chavez et al., 1996; Coorssen et al., 1996). In at least some polarized epithelial cells there are also two distinct types of constitutive vesicular carriers dedicated to delivery of newly synthesized proteins to the apical and basolateral domains, respectively, of the plasma membrane (Fig. 1) (Matter and Mellman, 1994; Ikonen et aI., 1995; Le Gall et aI., 1995). Moreover, in hepatocytes membrane and secretory proteins destined for the sinusoidal (basolatera1) domain are transported by separate vesicular carriers (Saucan and Palade, 1994). Recently, however, it has become clear that so-called "apical-cognate" and "basolateral-cognate" carrier vesicles are also present in nonpolarized as well as polarized cells (Yoshimori et al., 1996). Knowledge of the requirements for sorting of these various populations of vesicles, summarized below, is beginning to emerge but is still fairly rudimentary. Protein sorting and vesicular traffic in the Golgi apparatus 91

Figure 10. Consecutive serial sections of influenza virus-infected pancreatic B cells immunostained with anti• proinsulin (A) or anti-hemagglutinin (HA) (B) antibodies (protein A-gold technique). Proinsulin and HA labelling are present on the cisternae throughout the Golgi stack. Most of the proinsulin immunoreactivity in the transmost cisterna (arrows) is concentrated in the core of a condensing secretory granule (black arrowhead), while HA antibodies stain the membrane. Magnifications: (A, B) x 60,000; Bar=0.1 11m. From: Orci et al. (1987). 92 M.G. Farquhar and H.-P. Hauri

Sorting oflysosomal enzymes The best understood sorting mechanism is that worked out for sorting of lysosomal enzymes into clathrin-coated vesicles which is accomplished as follows. Lysosomal enzymes bind to MPRs somewhere in the Golgi stack; in the TGN the cytoplasmic tails of the receptors recognize and bind Golgi adaptor proteins which triggers assembly of clathrin coats on the vesicles (Glickman et al., 1989; Le Borgone et al., 1997), and the vesicles pinch offthe TGN thereby selectively remov• ing lysosomal enzymes from the secretory pathway. Coated vesicles carrying the acid hydrolases then fuse with the late and/or early endosome compartment where the low internal pH (pH 5.5) triggers dissociation of lysosomal enzymes from the receptor, and the receptors recycle to the GA. Two distinct MPRs have been identified - a cation-independent (CI-MPR) and cation-de• pendent (CD-MPR) receptor. The majority (70%) ofthe sorting at the TGN, as well as all the endo• cytic uptake of lysosomal enzymes, is done by CI-MPR; the CD-MPR functions mainly in trans• port of lysosomal enzymes from the TGN to endosomes (Glickman et aI., 1989; Kornfeld and Mellman, 1989). Distinction between the roles of the two MPRs at the TGN still remains unclear (Kornfeld and Mellman, 1989; Johnson and Kornfeld, 1992; Kornfeld, 1992; Mellman, 1996), but both are required. Neither receptor has an exclusive affinity for one or several lysosomal en• zymes (Pohlmann et aI., 1995) and a single type ofMPR, even when overexpressed, is not sufficient for targeting all lysosomal enzymes to lysosomes along the normal intracellular route (Kasper et aI., 1996). The mechanisms worked out for sorting of lysosomal enzymes into clathrin-coated vesicles via a M6P signal have been used as the paradigm to explain other sorting events, but to date no comparable receptor has been identified for any other Golgi-associated packaging event.

Sorting and packaging ofsecretory proteins into regulated secretory granules It is clear that in endocrine and exocrine glomerular cells with a regulated secretory pathway proteins are selectively sorted into dense-cored granules with the contents being highly concen• trated, almost crystalline in nature (Figs 2, 3 and 10). For example, in marnmotropes of the anteri• or pituitary gland, secretory proteins (consisting primarily of prolactin) were shown by quantita• tive EM autoradiography to be concentrated -200 times (Salpeter and Farquhar, 1981) within mature secretion granules. What is the mechanism involved? It has been known for some time that many secretory proteins, such as those of the exocrine and endocrine pancreas, anterior pituitary, and adrenal medulla have a tendency to aggregate in vitro, and aggregation is facilitated by mildly acidic pH and high calcium concentrations (Miller and Moore, 1990; Kelly, 1991; Colomer et aI., 1996). This has led to the hypothesis that sorting is initiated by spontaneous aggregation of regulated secretory proteins upon reaching the TGN (Kelly, 1985) which is assumed to be acidic (Glickman et aI., 1983; Anderson and Pathak, 1985). Some secretory prote• ins such as prolactin and insulin are capable of self-aggegation, whereas others may be incor- Protein sorting and vesicular traffic in the Golgi apparatus 93 porated into aggregates by piggy-back through binding to proteins such as the granins (chromo• granins and secretogranins) which are packaged into many endocrine and neuroendocrine secretory granules (Huttner et al., 1991). This scenario would still require interaction of the aggregates with putative receptors - i.e. specific Golgi or secretory granule membrane proteins • in order to trigger segregation into vesicles and budding from the Golgi. The fact that human growth hormone and insulin are sorted into the same regulated secretory granules suggests that there are common features on the aggregates and common membrane receptors that recognize different proteins (Burgess and Kelly, 1987; Moore et aI., 1988). A putative sorting signal and receptor have been identified for the sorting of proopiomelanocortin (POMC) to regulated secretory granules in pituitary cells. The sorting signal consists of an N-terminal amphipathic loop conformational motif (Cool et al., 1995), and the receptor consists of membrane-associated peptidasecarboxypeptidase E. It remains to be seen whether all prohormones and other proteins sorted to regulated secretory granules utilize the same mechanism (Cool et al., 1997).

Constitutive secretory vesicles: A default pathway? The mechanisms for the formation in the TGN of the post-Golgi vesicles that carry constitutively• secreted proteins and membrane proteins is poorly understood (Simon et aI., 1995). It has been assumed that in nonpolarized cells all proteins that lack targeting information are packaging into constitutive secretory vesicles considered a "default" pathway for delivery to the plasma mem• brane (Burgess and Kelly, 1987; Pfeffer and Rothman, 1987). There is as yet little evidence for this hypothesis. In fact what has been determined is that tyrosine-based targeting signals (see Tab. 2) are used for sorting into constitutive secretory vesicles at the TGN as well as at the cell surface (Matter and Mellman, 1994; Ktistakis and Roth, 1996; Ohno et aI., 1996). Tyrosine• based sorting signals in the cytoplasmic tail of MPRs and LAMPI/lgp120 have been shown to interact in vitro with API and AP2 adaptor proteins, implying that these proteins are sorted into clathrin-coated vesicles. Indeed by immunoelectron microscopy the two proteins colocalize in clathrin-coated structures budding from the TGN (Hunziker and Geuze, 1996). Hence these two proteins can reach the cell surface indirectly via endosomes. Likewise, transferrin receptor (putter et aI., 1995) and asialoglycoprotein receptor (Leitinger et al., 1995) are transported to the plasma membrane via endosomes, but it is not known if they are also packaged into clathrin-coated vesicles at the TGN. Clearly there is also an endosome-independent pathway to the cell surface that is followed by secretory proteins. Recently another population of TGN-derived vesicles, called p200-containing vesicles, which are distinct from both clathrin- and COP I-coated vesicles has been defined in NRK cells (Narula et aI., 1992). Besides p200, which was recently identified as nonmuscle myosin (A. Masch and E. Rodriguez-Boulan, personal communication; J. Stow, personal communication), the vesicles contain TGN38 (Wang et aI., 1995). The p200 protein has 94 M.G. Farquhar and H.-P. Hauri been shown to be essential for budding of these vesicles from the TGN in vitro (Wang et aI., 1995), suggesting that the p200 vesicles represent constitutive carriers destined for the plasma membrane. At present it is still not clear exactly how many distinct types of constitutive secretory vesicle populations bud from the TGN.

Sorting in polarized epithelial cells Studies in MDCK cells infected with enveloped viruses initially established that membrane proteins destined for the apical and basolateral plasma membrane domains (influenza hemagluti• nin HA and VSV-G protein, respectively) are sorted into different vesicular carriers in the TGN and delivered directly to the appropriate cell surface (Wandinger-Ness et aI., 1990; Ikonen et aI., 1995; Le Gall et aI., 1995). It was later found that in other cell types such as the hepatocyte, sorting into separate carriers occurs in the TGN, but delivery of apical plasma membrane proteins is indirect: they are delivered first to the basolateral cell surface where they are sorted and trans• ported to the apical domain by transcytosis (Bartles et aI., 1987; Schell et aI., 1992). Some cells, e.g. Caco-2 cells as well as intestinal epithelial cells in situ, make use of both the direct and the indirect routes (Louvard et al., 1992). How is sorting of apical and basolateral-directed vesicles by the TGN accomplished? It is now clear that basolateral targeting is a specific, signal-dependent process. Distinct signals have been identified in the cytoplasmic domains of membrane proteins that mediate sorting to the basolateral cell surface. Two such sorting signals have been defined - a tyrosine-based signal and di-leucine signal (Matter and Mellman, 1994; Ktistakis and Roth, 1996) which appear to be distinct signals that bind to distinct components. The tyrosine-dependent signal is similar to that found in endocytic receptors. In fact tyrosine-dependent signals involved in sorting into clathrin-coated pits, the TGN, lysosomes, and the basolateral surface of polarized cells are quite similar (see Table 2). If all these sorting events have the same generic signal, how is sorting at these locations carried out? Recent work suggests that the identity of residues surrounding the critical tyrosine residue, as well as the exact sequence of the tyrosine-based signal, are major determinants of selectivity in their interaction with adaptin complexes (Ohno et aI., 1996). Di-leucine signals are not nearly as common as tyrosine-based signals, but they have been described on several proteins (e.g. MPRs, T-cell antigen receptor y-chain and CD4, and have been shown to mediate many of the same processes as tyrosine-based signals including targeting to the basolateral surface of polarized epithelial cells, endosomal compartments and lysosomes (Matter and Mellman, 1994; Ktistakis and Roth, 1996). Many proteins with a di-leucine signal also contain a tyrosine-depen• dent signal but the interplay between the two signals is not understood. How are proteins sorted for apical delivery? Mechanisms of sorting of proteins directed to the apical plasma membrane are still poorly understood. Since removal of a basolateral targeting Protein sorting and vesicular traffic in the Golgi apparatus 95 signal leads to apical delivery and tail-minus mutants of basolateral proteins are transported to the apical surface, it has been suggested that apical delivery is by default. However, there is evidence that the lipid microenvironment of the TGN and glycosylation signals may be involved. The apical domain of many cells has an unusually high concentration of glycosphingolipids (GSLs) (Simons and van Meer, 1988), and these GSLs as well as membrane and secretory proteins are sorted and transported to the apical surface. It was suggested that the sorting of apically-destined proteins and lipids is connected and mediated by co-clustering of apical proteins and GSL• enriched microdomains in the TGN (Fiedler et aI., 1993). According to this hypothesis, glycolipid rafts form the nucleation site upon which apically-directed proteins and required machinery components assemble, leading to the exclusion of basolaterally destined cargo. It has been sug• gested that caveolin (VIP2l) through its cholesterol-binding properties promotes formation of protein oligomers and thereby facilitates microdomain formation (Zurzolo et aI., 1994; Murata et aI., 1995). These microdomains were isolated by virtue of their detergent insolubility and found to contain a specific subset of apically-directed proteins. Among them was VIP36 (Fiedler et aI., 1994) a protein that belongs to a new family of legume lectin homologues which includes ERGIC-53/p58. Based on the finding that VIP36 can bind 3H-galactose-Iabeled glycopeptides, it was proposed that VIP36 functions as a lectin in post-Golgi trafficking (Fiedler and Simons, 1995). The fact that glycosylated growth hormone is secreted preferably at the apical surface whereas nonglycosylated growth hormone is secreted from both sides lends credence to the idea that glycans playa role in protein sorting in the TGN (Scheiffele et aI., 1995). In summary, the mechanisms for apical delivery of membrane and secretory proteins are distinct from those involved in basolateral delivery and appear to be mediated by formation of GSL rafts and N• glycans.

Mechanism of vesicle budding, docking and fusion

Different transport vesicles - Common principles

Protein transport to and through the GA is mediated by distinct classes of transport vesicles. Three major types of vesicles defined by their coats have been characterized in detail to date • clathrin/adaptin-coated vesicles (CLA-CVs), COP I (coatomer)-coated vesicles (COP I-CVs), and COP II-coated vesicles (COP II-CVs). A number of other, less well characterized types of vesicles with connections to the GA have also been identified and will be discussed below (see Table 3). In broad terms there is a clear similarity in the mechanisms that drive budding, docking and fusion of all transport vesicles (reviewed in Rothman, 1994; Harter and Wieland, 1995; Kreis 96 M.G. Farquhar and H.-P. Hauri

Table 3. Coats and cargo receptors

Coat Loca- Coat Components Cargo Receptor Cargo Sorting Signal tion (recognized by (interacting with coat) coat)

Clathrin+ TGN ARFI CI-M6P-receptor M6-phosphate car- YKYSKV and LL Adaptins Clathrin Qteavy and light chain) VpslO boxypeptidase Y FYVF API (y-, Jjl-adaptin, ~I. (1) (via QRPL signal) PM Clathrin (heavy and light chain) many receptors hormones etc. Tyr-based motifs, An (a-, ~-adaptin, ~2, (2) VIP36* LL (LI) -KRFY COPI ERGIC ARFI ERGIC-53* ? -KKFF and coatOl~er (a-, ~-. ~'-, y-, p24* ? -KKXX or KXKXX Golgi 0-, £-, -COP) copn ER SarI ERGIC-53* Man-proteins RSQQE plus FF Sec23 complex emp24p/emv25* Gas Ip, invertase ? (Sec23p, Sec24p) Secl3 complex (SecI3p, sec31p) Lace-like TGN ARF?, rab6? TGN38* ? YQRL p200,p47 hTGN38* glycoproteins? ? VIP36* GalNAc-proteins? ? Caveolae PM CaveolinI,2IVIP2 I ? ?

* designation as a cargo receptor is putative

et ai., 1995; Salama and Schekman, 1995; Aridor and Balch, 1996; Bednarek et ai., 1996; Roth• man and Wieland, 1996; Schekman and Orci, 1996; Hauri and Schweizer, 1997; Robinson, 1997). Vesicle budding is initiated by the assembly of a protein coat from cytosolic components to the bud site of the donor membrane. Coat assembly is believed to drive vesicle budding and is followed by membrane fission releasing a fully formed coated vesicle. Included in the forming vesicle are cargo proteins, cargo receptors, and membrane proteins required for vesicle docking. Soon after budding the coat is lost, and the uncoated vesicle docks to an appropriate acceptor membrane. Pairs of integral vesicle and target membrane proteins ensure docking to the correct acceptor. Vesicular transport to the acceptor membrane may also involve motor driven guidance along rnicrotubules, particularly when it occurs over long distances, e.g. axonal transport (Kelly, 1990). Upon docking, additional proteins are recruited to the docking site, leading eventually to fusion of the vesicle with its target membrane. Subsequently, membrane components originating from the donor membrane, such as cargo receptors or docking receptors and lipids, must be recycled to the donor membrane by "retrograde" vesicular transport to maintain organelle integrity. The molecular details of vesicular formation, transport and docking are discussed in the next section. Protein sorting and vesicular traffic in the Golgi apparatus 97

Coat proteins and vesicle budding

Protein coats are thought to have a number of important functions in Golgi trafficking. They may (I) prevent mixing of compartments by protecting a donor membrane from forming tubules which fuse with neighboring membranes (Klausner et al., 1992); (2) serve as mechanical devices to sculpt a vesicle from a donor membrane (vesicle budding); (3) directly or indirectly recruit cargo proteins into the forming vesicle (sorting); (4) prevent a vesicle from premature fusion with its acceptor membrane; and (5) participate in regulating the accessibility of membrane receptors for microtubule-based motor proteins.

Clathrinladaptin-coated vesicles (CIA-CVs) CLA-CYs (Fig. 11), the first vesicles obtained in pure form (Pearse and Robinson, 1990), have become the paradigm for transport vesicles (Kirchhausen, 1993; Robinson, 1993; Schmid and Dahmke, 1995). They can bud either from the TGN (see Fig. 2) where they are involved in trans• port of lysosomal enzymes, or from the plasma membrane where they are involved in receptor mediated endocytosis. CLA coats assemble from clathrin triskelions (consisting of three 190 ill heavy and three -35 kD light chains) and heterotetrameric adaptor protein (AP) complexes (Traub, 1997). TGN-derived CLA-CYs contain AP1 complexes (consisting of'Y and ~ 1 adaptin, III and sl subunits) whereas plasma membrane-derived CLA-CYs contain the structurally related AP2 complexes (ex. and ~2 adaptin, 112 and s2 subunits). Vesicle formation is best understood in the case of plasma membrane CLA-CYs which is initi• ated by a two-step recruitment of coat proteins from the cytosol to an unknown saturable binding

Y C\IIthrin ------l V AP2s I )- _pI",. ,II I AP dodo. "lI III' ..... pn>leln? III o Oynomln 111

1UIIIIII'U"IIIIIUlIIIIUtlIIIIU"d,1 (®- - 00 00

Figure 11. The clathrin-coated vesicle cycle that drives receptor-mediated endocytosis. The following steps are depicted and described in detail in the text. Step 1: AP2 recruitment; step 2: clathrin assembly; step 3: dynamin recruitment; step 4: coated pit invagination; step 5: coated pit constriction requiring the redistribution of dynamin from the lattice and its assembly at the neck; step 6, coated vesicle budding, requiring a fusion event initiated on the external leaflet of the membrane. Figure provided by Dr. Sandra Schmid, Scripps Research Institute, La Jolla, CA. 98 M.G. Farquhar and H.-P. Hauri site on the plasma membrane. First, APs are recruited in an ATP- and GTP-independent reaction by a process that appears to be regulated by phosphorylation of adaptor subunits (Wilde and Brodsky, 1996). Bound APs then mediate the binding of clathrin triskelions to form a planar clathrin lattice on the cytoplasmic side of the plasma membrane, and the planar lattice invaginates to form a coated pit. The intrinsic capacity of CLA-coats to self-assemble in vitro in the absence of membranes suggests that the coat may provide the energy to drive membrane invagination, but direct evidence for this assumption is still lacking. It also remains unclear how a planar clathrin lattice consisting entirely of triskelion-containing hexagons can rearrange into a curved lattice consisting of both hexagons and pentagons, because such a process requires considerable mole• cular rearrangements (Liu et aI., 1995). Additional, still unknown cytosolic proteins and energy from ATP and GTP hydrolysis are required to complete CLA-CY budding. The GTPase dyna• min appears to playa key role in the fission process (Yallee and Okamoto, 1995; Warnock and Schmid, 1996; Urrutia et aI., 1997). In permeabilized synaptosomes treated with GTPyS, dyna• min assembles into dramatic rings and spirals forming a constriction around the elongated neck of coated pits (Takei et aI., 1995). GTP hydrolysis is thought to induce a conformational change in dynamin leading to neck squeezing followed by periplastic fusion and fission (Hinshaw and Schmid, 1995). Genetic evidence in Drosophila provides support for the primary role of dynamin in the fission process (Warnock and Schmid, 1996). In the temperature-sensitive, shibire ts-l mutant which has a defect in dynamin, coated pits are elongated and endocytosis is inhibited at the restrictive temperature. It is believed that the shibire ts-l dynamin can bind but not hydrolyse GTP. Uncoating of newly formed vesicles starts immediately: clathrin release is mediated by uncoating ATPase/hsc 70 in conjunction with the DnaJ homologue auxilin (Ungewickell et aI., 1995; Holstein et aI., 1996). Additional still unknown cytosolic factors are required to release the AP2 complex from the vesicles. Surprisingly, the process by which CLA-CYs are generated at the TGN is mechanistically more related to COP I-CY formation (see below) than to CLA-CY formation at the plasma membrane, in that for API to bind to the TGN it must be primed by the prior binding of ARF (Stamnes and Rothman, 1993; Traub et aI., 1993) which is not required in the case of CLA-CY formation. The latter was initially indicated by the observation that BFA, which blocks GDP/GTP exchange on ARF, inhibits CLA-CY budding from the TGN (Robinson and Kreis, 1992) but not from the plasma membrane. ARF is believed to mediate binding of the AP I complex to a putative docking receptor, and hydrolysis of GTP-ARF may lead to coat disassembly (Traub et aI., 1995). Thus far dynamin has not been directly implicated in vesicle fission from the TGN in mammalian cells, but a dynamin-like protein, Ypslp, required for the transport of enzymes from the GA to a preva• cuolar compartment, has been identified in yeast (Conibear and Stevens, 1995; Notwehr et aI., 1995). Protein sorting and vesicular traffic in the Golgi apparatus 99

Clathrin/adaptin coats forming at the plasma membrane recruit cargo by binding to specific signals located on the cytoplasmic tails of cargo receptors. The best characterized CLA-coated pit targeting signal is the tyrosine-containing signal (Tab. 2) first described in the LDL receptor and later found in a number of receptors (transferrin, EGF, and MPRs) as well as the TGN protein TGN38, and the lysosomal membrane protein LAMP-l (Trowbridge et aI., 1993; Ktistakis and Roth, 1996). The cytoplasmic tails of several cell surface receptors interact specifically with the AP2 adaptor complex through their tyrosine-containing localization signals (Boll et aI., 1996). The cytoplasmic domains of LgplLAMPS and TGN38, which undergo dual sorting at the TGN and the plasma membrane, bind to both APl- and AP2 adaptor complexes but with different affinities (Ohno et aI., 1995; Ohno et aI., 1996). Another such sorting signal found on several proteins (both MPRs, T-cell receptor ychain, CD4) is a di-leucine (or leucine-isoleucine) peptide. Both the tyrosine and di-leucine signal-based targeting mechanisms use distinct saturable compo• nents, but the binding partner for the di-leucine signal is unknown (Marks et al., 1996). Specific adaptin-receptors have been proposed to exist (Mahaffey et aI., 1990; Robinson, 1993). Synaptotagmin I, a calcium-sensor during synaptic vesicle exocytosis in the brain (see paragraph on vesicle docking and fusion) was reported to bind AP2 complexes with high affinity in vitro (Zhang et aI., 1994). Conceivably, a basic level of CLA-CY formation may be mediated by specific adaptin-binding proteins whereas increased receptor levels or ligand-induced receptor clustering can enhance this process by providing more adaptin binding sites.

COP I-coated vesicles COP I-coats (Fig. 12) have been implicated in many different traffic pathways including exit from the ER (Peter et aI., 1993; Bednarek et aI., 1995), ERGIC-to-Golgi (Pepperkok et al., 1993), Golgi-to-ER (Cosson and Letoumeur, 1994; Letoumeur et aI., 1994; Lewis and Pelham, 1996), through the Golgi (Rothman and Orci, 1992), and early-to-Iate endosomes (Whitney et aI., 1995; Aniento et aI., 1996). The coat of COP I-CYs consists of the small GTPase ARF1 and coatamer, a -700 kD complex of seven coat (COP) proteins (a, ~, W, y, 0, E, s) (Duden et aI., 1991b; Waters et aI., 1991; Kreis and Pepperkok, 1994). Interestingly, ~, 0 and SCOP display limited sequence homology with the ~, I.l and cr adaptin subunits, respectively, of CLA-CYs (Duden et al., 1991 b; Serafini et aI., 1991; Cosson et aI., 1996; Faulstich et aI., 1996), suggesting that the two sets of coat proteins arose from a common ancestor. The GDP-bound form of ARF1 and the coatomer complex exist separately in the cytosol. Budding of COP I-CYs is initiated by the recruitment of GDP-ARF1 to putative ARF receptors on the donor membrane (ERGIC or Golgi) (Fig. 12) where it is activated and converted to its GTP-bound conformation by a membrane• associated GTP-GDP exchange factor (GEF) (Donaldson et aI., 1992; Helms and Rothman, 1992) resulting in membrane attachment of ARFI via an as yet unidentified receptor. ARF-GEFs 100 M.G. Farquhar and H.-P. Hauri

COP -Coat Asse by o tomer I. ~ (

Figure 12. Hypothetical model for the stepwise assembly of the COP I coat from information obtained in both mammalian cells and yeast. The proposed steps are based upon the mechanism of c1athrin-coated vesicle formation. Step I: COP I formation on the endoplasmic reticulum (ER) and GA is initiated by activation and membrane recruitment of the small GTP-binding protein ARF. Activation of ARF is catalyzed by a putative GDP-GTP exchange factor (GEF) that is attached in a brefeldin A (BFA)-sensitive manner to the target organelle through a putative linker protein (GEF-AP). The interaction of ARF-GTP with the membrane is enhanced by an N-terminal myristate chain. Membrane-bound ARF stimulates the local modification of lipids by acting upon phospholi• pase D (PLD). The negatively charged lipids (indicated by the blue circle) and the cytosolic domains of cargo proteins and v-SNAREs cooperate to form a high-affinity coatomer-binding site. Upon binding, coatomer induces the clustering of these proteins (step II) and creates a curved membrane (step III), a process that leads ultimately to the release of a coated vesicle. From: Bednarek et aI., 1996. Abbreviations: GDP: guanosine diphosphate; GTP: guanosine triphosphate; v-SNARE: vesicle-bound soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor. Protein sorting and vesicular traffic in the Golgi apparatus 101 have been identified in yeast and mammals, and all exhibit partial sequence homology with yeast Sec7p (Chardin et al., 1996; Morinaga et aI., 1996; Peyroche et aI., 1996). ARFI binding to the membrane triggers binding of coatomer to ARFI via the P-COP subunit (Zhao et al., 1996) and budding occurs (Orci et aI., 1993; Hara-Kuge et aI., 1994). Precisely how ARF functions is not yet clear, but recent evidence suggests that by activation of phospholipase D (PLD) (which hydrolyses phosphatidylcholine to phosphatidic acid and choline) it may generate a negatively charged membrane site to which coat proteins attach noncovalently (Ktistakis et aI., 1996). Such a local lipid change, rather than the coat itself, may be the initial driving force for budding. In line with a compositional change of the lipid bilayer during budding is the morphological finding (Orci et aI., 1996) that the interleaflet clear space is reduced in the membrane of COP 1- and COP II-coated buds on vesicles when compared to nonbudding areas of the same membrane. Although coatomer and ARFI are the only cytosolic proteins required to produce COP I-CY from isolated Golgi membranes in a cell-free system (Orci et aI., 1993), additional factors are likely to be required in vivo to define the correct site of budding and to regulate the efficiency of budding. It is known that subsequent vesicle fission requires palmitoyl-coenzyme A (Ostermann et aI., 1993), perhaps reflecting a palmitoylation event. Palmitoylation may be needed to activate a constriction protein or a periplastic fusion factor operating at the neck of the coated bud. After budding, hydrolysis of GTP bound to ARFI and release of GDP-ARFI triggers coat disas• sembly, a process inhibitable by GTPyS or AlF4- (Melancon et al., 1987). How is cargo selectively packaged into COP I-CYs? Different mechansims can be envisioned but in the simplest case, as with CLA-CYs, selection of cargo is coupled to coat binding. Indeed, as mentioned earlier, in vitro experiments have shown that the di-Iysine ER retrieval motif of transmembrane proteins can directly bind coatomer (Cosson and Letoumeur, 1994), and yeast coatomer mutants are defective in ER retrieval of reporter proteins tagged with this motif (Gaynor et al., 1994; Letoumeur et al., 1994; Townsley and Pelham, 1994; Cosson et aI., 1996), suggesting that retrieval of di-Iysine-bearing ER proteins is mediated directly by the interaction of cargo and coatomer. Which coatomer subunit recognizes the di-Iysine motif is still a matter of debate (see Lowe and Kreis, 1995; Harter et al., 1996). The demonstration of the direct interaction between coatomer and the di-Iysine motif led to the single pathway model whereby COP I-CYs are assumed to be involved only in retrograde trans• port from the Golgi back to the ER (Pelham, 1994; Lewis and Pelham, 1996). According to this model all effects of coatomer mutants on anterograde pathways in vivo would be secondary to a retrieval defect because anterograde and retrograde transport between two organelles is highly interdependent. Since COP I is associated with a least four compartments (ER, ERGIC, GA, endosomes), the single pathway model would also predict that some of the localizations of COP I 102 M.G. Farquhar and H.-P. Hauri in the cell reflect COP I functions other than vesicle budding, such as organelle maintenance (Duden et al., 1991a). The other view is that more than one traffic route is mediated by COP I-CYs. If this is the case, how can coatomer fulfill different tasks? It is unlikely that the many demands for coatomer in the cell are met by multiple closely related isoforms of coatomer subunits because there is only one copy of each coatomer subunit per haploid genome in yeast (Bednarek et aI., 1996). However, the recent identification of a novel mammalian 1;-COP-related protein, termed 1;2-COP (Whitney and Kreis, 1996), suggests the existence of pathways not present in yeast. Alternative possibilities for specificity would be (I) the existence of different coatomer subcomplexes as reported for endo• some-associated COPs (Whitney et aI., 1995; Aniento et aI., 1996), or (2) different post• translational modifications of coatomer subunits such as phosphorylation (Sheff et aI., 1996) or other conformational switching mechanisms by which coatomer can recognize di-Iysine and other putative signals (Fiedler et aI., 1996). It is fair to state that none of the current hypotheses on the role of COP I can easily accommodate all the observations.

COP II-coated vesicles (COP II-CVs) A combined genetic and biochemical approach in yeast identified a distinctive set of proteins that promote vesicle budding from the ER in vitro (Barlow et aI., 1994; see Duden and Schekman chapter). These vesicles, designated COP II-coated vesicles, have a life cycle very similar to that of COP I-CYs (Fig. 13). Budding starts with the recruitment of an ARF-related small GTPase, Sarlp, to the ER membrane and conversion to its GTP-bound form by a GEF (Sec12p). This triggers vesicle budding by the attachment of the coat protein complexes, Sec23p and Sec13p, which are unrelated to coatomer. Sec23p itself has Sarl-specific GAP activity leading to coat disassembly. No palmitoyl-coenzyme A is required to form a COP II-coated vesicle. Mammalian homologues of Sarlp, Secl3p, and Sec23p have been identified and their localization in rough ER, transitional elements of the rough ER, and nearby vesicles is consistent with the notion that COP II-CYs mediate anterograde protein transport from ER to ERGIC (Orci et aI., 1991; Aridor et aI., 1995; Shaywitz et aI., 1995; Tang et aI., 1997). There is no indication of involvement of COP II-CYs in other vesicular steps. How are cargo proteins selected and packaged into COP II-CYs? As with CLA-CYs and COP I-CYs, a mechanism by which the coat selects cargo is the most likely scenario. A candidate protein linking cargo to coats is the mannose-selective lectin ERGIC-53/p58 that recycles in the early secretory pathway (Saraste et aI., 1987; Schweizer et aI., 1988; Schweizer et aI., 1990; Saraste and Svensson, 1991; Schindler et aI., 1993; Itin et aI., 1995a; Itin et aI., 1995b; Itin et aI., 1996). The cytoplasmic domain carries both an (anterograde) ER export determinant and a (retrograde) di-Iysine ER retrieval signal. In a bead-binding assay peptides corresponding to the Protein sorting and vesicular traffic in the Golgi apparatus 103

COPII -Coat Assembly

)~GDP II. ~TP

Figure 13. Hypothetical model for the stepwise assembly of the COP II coat based mainly on studies in yeast. Step I: Sarlp binding to the ER membrane is facilitated by Secl2p-mediated nucleotide exchange. Genetic studies imply a functional link between Sarlp, Sed4p and Sec16p (Gimeno et aI., 1995). Binding of Sarlp to the mem• brane may be stabilized by interaction with Sed4p and Secl6p. This complex then serves as a docking site for two protein complexes, Sec23p-Sec24p and Secl3p-Sec31p. Sec23p-Sec24p recognizes sorting motifs on cargo and targeting proteins, marking them for inclusion into vesicles. Although there is no direct evidence that the COP II components bind to cargo and v-SNAREs, it is likely that this step occurs, at least transiently, to facilitate the selective packaging of cargo into vesicles. Step II: Polymerization of the Sec 13p-Sec31p complex serves to cluster activated cargo and targeting molecules. Interaction between Sed4p, Sec16p and the cytosolic COP II components is likely to be transient because Sed4p is not found in ER-derived vesicles. GTP hydrolysis may discharge Sar1p from activated COP-coat-membrane-cargo complexes and promote recycling of the GTP-binding protein. Step III: Additional binding of the Sec23p-Sec24p and Sec 13p-Sec31 p complexes induces membrane curvature and leads to vesicle release. From: Bednarek et al. (1996). 104 M.G. Farquhar and H.-P. Hauri cytoplasmic domain of ERGlC-53 can bind both the COP II coat protein Sec23p and coatomer (COP I). Inactivation of the export determinant abolishes Sec23p binding, and inactivation of the di-Iysine motifabolishes coatomer binding (Kappler and Hauri, unpublished observation). These binding features suggest that ERGIC-53 functions as a transport receptor that may facilitate anterograde transport ofnewly-synthesized glycoproteins by actively recruiting them into COP II• CVs during budding from the ER. After dissociation of cargo in post-ER compartments, empty ERGlC-53 would be recycled in COP I-CVs. A candidate transport receptor in yeast is Emp24p. Deletion of Emp24 renders transport of a subset of soluble secretory proteins inefficient (Schim• moiler et aI., 1995), but it is unknown if Emp24p directly binds these cargo proteins. Emp24 is the founding member of a class of related 24 kD proteins that may operate as cargo receptors for various transport steps (Stamnes et aI., 1995; Belden and Barlowe, 1996; Sohn et aI., 1996). While different members of the p24 family can bind different sets of COP I coat proteins in vitro (Fiedler et aI., 1996), the suggestion that they operate as sorting receptors remains to be proven.

Vesicle docking andfusion

A conceptual framework for explaining how transport vesicles dock to an acceptor membrane was formulated in the SNARE (Soluble NSF Attachment Protein Receptor) hypothesis (Sollner et aI., 1993b). The SNARE hypothesis states that the specificity of vesicle targeting is generated by complexes that form between membrane proteins on the transport vesicle (v-SNARE) and membrane proteins on the acceptor membrane (t-SNARE). The SNARE complex forms a scaf• fold for the sequential recruitment from the cytosol of the general trafficking factors NSF (NEM• sensitive factor; Sec18p in yeast) and a-SNAP (soluble NSF attachment protein a; Secl7p in yeast) that induce fusion in conjunction with accessory factors. The hypothesis is based on the finding that NSF and a-SNAP form specific complexes with three membrane proteins from crude Triton Xl00-solubilized bovine brain membranes. One pro• tein was identified as the synaptic vesicle protein VAMP (also known as synaptobrevin) and the other two as the presynaptic plasma membrane proteins, syntaxin 1 and SNAP-25 (synaptosome• associated protein of 25 kD; no relation to a-SNAP). Hence VAMP was termed a v-SNARE, and syntaxin 1 and SNAP-25 were termed t-SNARES. Similar v-SNARE/ t-SNARE pairs are now known for various pathways in animal and yeast cells (Tab. 4), but it is not known if SNAREs are the only integral membrane proteins required for vesicular targeting and fusion (Stidhof, 1995; Ting and Scheller, 1995; Rothman and Wieland, 1996). The mechanism of docking and fusion is most completely understood for the vesicles that connect ER to ERGIC (or GA in yeast) and GA or synaptic vesicles to the plasma membrane ao

~. Table 4, SNAREs and accessory factors ::l gen Jg' Accessory Factors § Pathway Organism v-SNARE t-SNARE Rab Secl-like Other 0-

2 ~ Synapse mammals VAMP (=synaptobrevin) syntaxinI Rab3p muncl81 nseclp synaptotagmin (Ca + sensor) o' SNAP25 ephexin c:: synaptophysin (protects VAMP) i!f q ER toGA yeast Bosl Sed5 (syntaxin5-like) YPTlp Slylp Bet3 8l Bet! Sec34p, Sec35 (interact with YptIp) (') 5' Sec22p Usolp (may promote docking) Ykt6/p26 Sec?p 1f Yp26 ~ ER to ERGICI GA mammals rBet! syntaxin5 RabI rSlyIp pII5 (homolog of Usolp) € rSec22 :8 p28 ~ GA toER yeast Ufel Sec2OpITip20p complex en

Intra-GA (retrograde) yeast Sftl Sed5

GA to PM yeast Sncll2 (synaptobrevin-like) Ss01l2 (syntaxin-like) Sec4p Sec I Sec6p/Sec18p/Sec25p-complex Sec9p (SNAP25-like)

GA to endosome yeast Pepl2 (syntaxin-like) Vps2Ip Vps45p Slpl Pep? (interacts with Vps45p)

Glut4-compartment to PM mammals VAMP2 syntaxin4 Rab4 muncl8c

...... o VI 106 M.G. Farquhar and H.-P. Hauri

(Ferro-Novick and Jahn, 1994; Rothman, 1994; Bennett, 1995; Novick et al., 1995; Siidhof, 1995; Ting and Scheller, 1995; Pfeffer, 1996; Rothman and Wieland, 1996). Every compartment expresses its own pathway-specific v-SNAREs, for instance Bos1p, Bet1p (Ferro-Novick and Jahn, 1994) and Sec22p (Newman et aI., 1992) in the case of the ER, that are packaged into transport vesicles during budding. The acceptor membrane possesses specific t-SNAREs, for instance syntaxin 5 in the case of ERGIC (Banfield et al., 1994; Dascher et aI., 1994), that are required for the vesicle docking. Pairing of v- and t-SNARES is specific and assumed to occur via a-helical coiled coil regions present in the cytoplasmic domain of both types of receptor proteins. SNAREs belong to the class of C-terminally anchored proteins and are therefore opti• mally positioned to interact pairwise (Pelham et al., 1995). More than one type of v-SNARE can interact with a given t-SNARE, as exemplified by the fact that in a yeast in vitro system, the ER• derived v-SNAREs, Bos1p, Bet 1p, Sec22 and YKT6/p26, pair with the Golgi t-SNARE Sed5p (Sogaard et aI., 1994). Multiple SNARE interactions have also been described to occur at the synapse where the synaptic v-SNAREs, synaptobrevin and synaptotagmin, interact with two plasma membrane t-SNARES, syntaxin1 and SNAP-25 (Schiavo et al., 1997). Pairing of v- and t-SNAREs in the cell is regulated by accessory proteins (Tab. 4), most notab• ly by Rab GTPases (Ypt GTPases in yeast) and Sec1-related proteins. Rab proteins facilitate SNARE complex formation as indicated by the observation that v-SNARE/t-SNARE interaction in a yeast in vitro system does not occur in the absence of functional Ypt1p (Sogaard et aI., 1994). Recent evidence from studies on Rab5 suggests that the GTP hydrolysis of Rab proteins acts as a timer that determines the frequency of membrane docking/fusion events (Rybin et aI., 1996). Whether other rabs have a similar timer function is uncertain because, unlike rab5, most other rabs have a slow GTPase activity. Sec1 proteins act as t-SNARE protectors. They bind to specific t-SNAREs and thereby prevent their assembly (Novick et aI., 1995). In this way they prevent nonproductive t-SNARE/t-SNARE interaction in the acceptor membrane. The exact sequence of events of docking and fusion is not entirely clear yet. Whether the SNARE pairing is the first event of docking is currently debated, based on the finding that neuronal t-SNAREs are not restricted to the sites where synaptic vesicles dock and fuse (see Siidhof, 1995; Rothman and Wieland, 1996, for different views). Moreover, because t-SNAREs are protected by SecI proteins, they need activation to allow for assembly with their cognate v• SNARE. A possibility is that Rab proteins on transport vesicles recruit accessory docking fac• tors. These docking factors or the Rab protein could then free the t-SNARE, thus allowing v• SNARE/t-SNARE assembly (Scheller, 1995; Pfeffer, 1996). Candidate docking factors acting in conjunction with Rab proteins are the Sec6p/Sec8p/Sec15p complex required for vesicle docking to the plasma membrane in yeast (TerBusch and Novick, 1995), pl15 (Uso1p in yeast) required for ER-Go1gi transport (Sapperstein et aI., 1996), and the rabaptin 5 complex involved in endoso- Protein sorting and vesicular traffic in the Golgi apparatus 107 mal docking events (Stenmark et aI., 1995). Once the SNARE complex is formed, NSF and SNAP proteins assemble at the docking site. Then NSF dissociates the SNARE complex in an ATP-dependent process, the SNAPs dissociate and fusion occurs. Whether the NSF ATPase reaction directly induces bilayer fusion without the involvement of other proteins remains to be clarified. Recent evidence from studies on homotypic fusion of yeast vacuoles suggest that (X.• SNAP and NSF may function in a predocking stage rather than in the fusion process (Mayer et aI., 1996). However, it is possible that this inverse scenario is true only for homotypic fusion events. Table 4 gives an overview of SNARE-dependent vesicular pathways. Remarkably, the SNARE hypothesis applies to both constitutive and regulated secretion. Yet there appears to be at least one exception. Studies on the molecular requirements of apical transport of influenza hemagglutinin from GA to plasma membrane in (permeabilized) polarized MDCK cells revealed that this path• way was independent of NSF, (X.-SNAP, Rab GTPase activity and a v-SNARE (tested by a v-SNARE inactivating neurotoxin) (Ikonen et aI., 1995). The data point to a SNARE-independent mechanism for apical vesicle docking and fusion. Instead it was proposed that theCa2+ -depen• dent phospholipid-binding protein, annexin 13b, may play a role in this process because anti• bodies to annexin 13b specifically inhibit apical transport (Fiedler et al., 1995). Alternatively, it is possible that SNAREs are nevertheless involved in apical docking but fusion would occur in a NSF/SNAP-independent manner. Such a mechanism would explain the finding that overexpres• sion of the apically-localized putative t-SNARE, syntaxin 3, inhibits direct transport from GA to apical plasma membrane of polyimmunoglobulin-receptor mutants (Weimbs et al., 1996). Alter• natively, two different apical pathways may connect GA and plasma membrane, only one of which may exhibit the classical SNARE features. Anterograde transport through the Golgi stack has been proposed to occur by multiple vesicular steps (Rothman and Orci, 1992). Surprisingly, no single Golgi v-/t-SNARE pair has been identi• fied yet for this traffic route. Even though more than 30 Rabs have been identified only a single Rab, Rab6, is known to be associated with the stacked GA (Goud et aI., 1990; Anthony et aI., 1992; Jones et al., 1993; Martinez et al., 1994). Moreover, sequencing of the yeast genome has not revealed candidate t-SNAREs for antero• grade intra-Golgi transport. Unlike v-SNAREs, t-SNAREs are homologous to such an extent that they can be identified by genome analysis. This raises the question of whether anterograde transport through the GA is mediated by vesicles that do not fit into the SNARE hypothesis or whether anterograde transport could be nonvesicular, occurring by cisternal progression. Defini• tive answers to these important questions are not yet at hand. 108 M.G. Farquhar and H.-P. Hauri

Less well-characterized transport vesicles

Can CLA-CYs, COP I-CYs, COP II-CYs serve as paradigms for all other transport vesicles? Although likely, this question cannot be answered with certainty because the characterization of other vesicle types is at an early stage.

Constitutive exocytic transportfrom the TGN to the plasma membrane Neither clathrin nor coatomer is involved in this process, but, as mentioned earlier, a novel lace• like coat was identified on TGN membranes of nonpolarized cells (NRK) by electron microsopy that may drive vesicle budding (Ladinsky et aI., 1994). Biochemical experiments have implicated at least five proteins in the formation of constitutive exocytic vesicles from the TGN: a dimer of TGN38 (a putative transport receptor recycling between TGN and plasma membrane (Stanley and Howell, 1993; Kain et aI., 1996), a 62 kD Serffhr kinase associated with small GTPases (including Rab6), an = 100 kD catalytic subunit of a pI3-kinase (Wang et aI., 1995), and p200 that has coat-like properties (Narula and Stow, 1995). The fact that antibodies against the cytoplasmic domain of TGN38 or against p200 inhibited transport to the cell surface when electroporated into NRK cells provides evidence that these proteins are essential for this pathway (Wang et aI., 1995).

Transport from the TGN to the apical and basolateral domains ofthe plasma membrane in polarized epithelial cells As discussed above, the apical pathway may not conform to the SNARE hypothesis, but the basolateral does (Ikonen et aI., 1995). Simon et aI. (1996a, b) developed a system that allows in vitro generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which YSY-G (a basolaterally targeted protein) is allowed to accumulate in the TGN at 20°C. Vesicle formation requires activation of an ARF GTPase but unlike COP I-CYs, GTPyS promotes rather than prevents vesicle formation. By electron microscopy, TGN-derived vesicles and COP-CYs are indistinguishable. These basolaterally-directed vesicles appear to be different from the TGN38-containing vesicles described by Wang et al. (1995).

Vesicles involved in the recycling ofmannose 6-phosphate receptors (MPRs)from the late endosome to the TGN Docking and fusion of these recycling vesicles appears to occur by a SNARE mechanism because the process is dependent on Rab9 complexed with GDI (Lombardi et aI., 1993; Dirac• Svejstrup et aI., 1994) as well as NSF and a-SNAP (Itin, c., personal communication). Nothing is known about the mechanism of budding of these vesicles except that clathrin does not seem to Protein sorting and vesicular traffic in the Golgi apparatus 109 be involved (Draper et a!., 1990). Perhaps the recently discovered adaptor-like complex, now called AP3 (Simpson et aI., 1996; DeU' Angelicaet aI., 1997), is involved in this pathway.

Vesicles that mediate recycling ofplasma membrane proteinsfrom early endosomes to the plasma membrane This class of vesicles includes constitutively recycling vesicles for receptors, such as LDL and transferrin receptors, present in aU ceUs, and ceU type-specific recyling vesicles such as synaptic vesicles of neurons and neuroendocrine ceUs, Glut4-vesicles in adipocytes and muscle ceUs, and MHC class II-containing vesicles in antigen-presenting ceUs (MeUman, 1996). Best characterized is the life cycle of synaptic vesicles (KeUy, 1993; Siidhof, 1995; De CarniUi and Takei, 1996) particularly in terms of mechanisms of docking and fusion (ScheIIer, 1995, see Table 4).

Caveolae A special class of vesicles are caveolae (initially termed plasmalemmal vesicles: Palade, 1953). Caveolae form at the plasma membrane of most ceIIs. They are defined by the coat protein cave• olin of which three isoforms are known (Rothberg et aI., 1992; Parton, 1996). Caveolin (VIP21) was independently discovered as a major component ofTGN-derived apical transport vesicles in epithelial ceIIs and was highly enriched in detergent-insoluble, glycosphingolipid-enriched microdomains (Kurzchalia et a!., 1992; Dupree et aI., 1993; Fiedler et aI., 1993). The function of caveolae is currently a matter of considerable debate. Diverse functions have been proposed, including transcytosis in endothelial ceIIs, where they serve to transport adhesion and other mole• cules across the endothelium (Ghitescu et a!., 1986; Predescu et a!., 1994; Palade, 1997; Predescu et a!., 1997), receptor-mediated uptake of smaII molecules (potocytosis: Rothberg et a!., 1990; Anderson et a!., 1992), alternative endocytosis (Montesano et aI., 1982; Simionescu et aI., 1982), signal transduction (Sargiacomo et a!., 1993; Lisanti et a!., 1994), calcium uptake (Fujimoto, 1993) and polarized sorting to the apical domain of epithelial ceIIs (Kurzchalia and Parton, 1996; Parton, 1996). Two major issues are of great interest. First, are caveolae in vivo permanently attached to the plasma membrane or can they detach and fuse with the endomembrane system, as indicated by work on the capiIIary endothelium cited above and as seen in okadaic acid-treated ceIIs (Parton et a!., 1994). The second, even more chaIIenging issue is to clarify the unusual recycling itinerary reported for caveolin. In fibroblasts it was found to detach from plasma membrane-attached caveolae, to translocate into the lumen of the ER, and to recycle to the plasma membrane via ERGIC and the GA (Smart et a!., 1994; Conrad et a!., 1995). Ifconfirmed, these findings on caveolin recycling suggest a novel, unconventional cycling pathway between plasma membrane, ER and GA. It wiII also be interesting to know if caveolae conform to the SNARE hypothesis of docking and fusion. In view of the apparent late addition of caveolae during euka- 110 M.G. Farquhar and H.-P. Hauri ryotic evolution (caveolin has not been found in yeast, K. Fiedler, personal communication) and the fact that some mammalian cells lack identifiable caveolae, it is interesting that a protein immunologically related to the v-SNARE, VAMP-2, has been reported to be enriched in caveolar fractions from rat lung (Schnitzer et al., 1995; Schnitzer et al., 1996). This may suggest either that caveolae docking and fusion involves a SNARE-type mechanism or, alternatively, that v-SNARE enrichment in caveolae simply reflects an inactive v-SNARE that recycles via caveolae.

Regulation ofmembrane traffic by small GTPases, heterotrimeric GTPases, and phosphoinositides

Maintenance and adaptation of bidirectional transport without compromising structural and func• tional organelle integrity requires sophisticated mechanisms of regulation. Growing evidence suggests that key components controlling signal transduction at the cell surface are also critically involved in regulating distinct steps of membrane traffic to and through the GA. Major roles are played by small GTPases of the ras superfamily, heterotrimeric G proteins and phosphoinositide lipids. Crosstalk between these signalling molecules is an important feature of traffic regulation. Regulation can be envisioned to occur at different steps of vesicular transport including coat assembly, coat-mediated cargo sorting, vesicle budding, vesicle fission, uncoating, vesicle interac• tion with the cytoskeleton, and vesicle docking and fusion. GTPases are well suited to regulate macromolecular interactions accompanying these processes because they act as molecular switches by alternating between the GDP- and GTP-bound forms. Among the GTPases, those that are most relevant for membrane traffic are (1) several small GTPases of the ras superfamily including ARF/Sar, Rab (Ypt), and Rho (including Rho, Rac and CDC-42), (2) the dynamins, and (3) the heterotrimeric G proteins (Nuoffer and Balch, 1994). Phosphoinositides can either recruit or activate proteins essential at various traffic stations by undergoing phosphorylation and dephosphorylation of the inositol ring (Liscovitch and Cantley, 1995; De Camilli et aI., 1996).

Small GTPases The role of the small GTPases ARFI and SarI in budding, and Rab (Ypt) in docking, have al• ready been discussed. There are at least six different ARF proteins designated ARFI to 6 (Donald• son and Klausner, 1994) that have different but overlapping distributions. Besides ARFl, ARF6 is the next best characterized protein. It appears to playa role in regulating the endocytic pathway (Peters et aI., 1995), perhaps by interacting with heterotrimeric G proteins (Colombo et al., 1995). Rab GTPases represent a family of over 30 proteins that are localized to different vesicular and organellar membranes (Chavrier et aI., 1990; Ferro-Novick and Novick, 1993; Novick and Protein sorting and vesicular traffic in the Golgi apparatus III

Brennwald, 1993; Zerial and Stenmark, 1993; Nuoffer and Balch, 1994; Pfeffer, 1994; see Fig. 14). They are believed to exercise a proof-reading function, checking that each vesicle fuses with the appropriate target. Originally it was believed that each Rab is dedicated to a specific step in transport. However, we now have more Rabs than defined steps, as there are less than 30 relays of transport carriers. To date several tissue- or cell-type specific Rabs have been identified. Also, multiple Rab proteins may be associated with the same carrier vesicles and function together. In support of this idea, at least five different Rabs are associated with the transcytotic pathway (Jin et aI., 1996), at least seven Rabs with the zymogen granule membrane (Lambert et al., 1990; Padfield and Jamieson, 1991; Schnefel et aI., 1992), and six Rabs with endosomes (Chavrier et a!., 1990; Van Der Sluijs et al., 1991; Lutcke et al., 1994; Weber et al., 1994). Conversely, the same Rab can be involved in more than one vesicle pathway. Such Rabs may act as more general factors in the transport process. Rho GTPases (A, B, and C) play dynamic roles in the regulation of the actin cytoskeleton, thereby controlling cell adhesion, motility and shape, but they also participate in traffic control (see the chapter by Kreis, this volume). Rho can stimulate regulated secretion in mast cells (Price et al., 1995) and clathrin-independent endocytosis in Xenopus (Schmalzing et aI., 1995), and it inhibits clathrin-mediated endocytosis in mammalian cells (Lamaze and Schmid, 1995). Another family member, RhoD, causes rearrangement of the actin cytoskeleton and governs early endosome dynamics and distribution (Murphy et a!., 1996), suggesting that Rho GTPases may provide a molecular link between membrane traffic and the actin cytoskeleton. Rho proteins have not been localized to the GA yet, but given the observation that both ARFI and Rho can activate the same isoform of Golgi-associated phospholipase D (Ktistakis et aI., 1995) we predict that Rho may also have a regulatory function in the GA. Interestingly, in vitro ARF and Rho activate the same purified PLD isoform, PLDI. PLD1 can also be activated by protein kinase Cn in a kinase-independent manner. ARF, Rho, and PKC activate PLD1 synergistically, suggesting that they interact with different sites on the enzyme (Hammond et a!., 1997). Because phosphatidic acid, one of the products of the PLD action, can itself act as an intracellular modulator of Raf and certain PKC isoforms (Ghosh et a!., 1996), or can be metabolized to generate the second messengers diacylglycerol, arachidonic acid and lysophosphatidic acid (Klein et aI., 1995), ARF and Rho can be viewed as members of signal transduction cascades that converge on PLD. PLD may therefore act as an integrative regulator of ARF- and Rho-mediated effects. It has been proposed that the activation of PLD1 by ARF, Rho and PKC links the process of vesicle formation to activation of cell surface receptors (Frohman and Morris, 1996). Alternatively, such receptors may reside on Golgi membranes, where they could transduce signals from the lumen to the cytoplasm in response to interaction with cargo proteins. Indirect evidence for such a mechanism comes from the finding that the formation of 112 M.G. Farquhar and H.-P. Hauri

rab10 rab12 Goigi

Lysosome

Figure 14. Intracellular localization of some ubiquitously expressed Rab proteins. Rabla, Rablb, and Rab2 have been localized to and function in transport between the endoplasmic reticulum and GA. Rab6, Rab8, and Rab12 are associated with the GA. Rab6 is distributed from medial-Golgi to the trans-Golgi network Rab8 is localized to the trans-Golgi network, post-Golgi vesicles and the plasma membrane and regulates traffic between these compartments. In the endocytic pathway, Rab5a is associated with the plasma membrane, clathrin-coated vesicles and early endosomes and regulates endocytic transport, whereas Rab4a, which is present on early endosomes, plays a role in recycling from early endosomes to the cell surface. Rab5b, Rab5c, and Rab4b appear to share the functional properties of Rab5a and Rab4a, respectively. Rab7 and Rab9 are both associated with late endosomes, but only Rab9 controls transport to the trans-Golgi network. Rab18, Rab20, and Rab22 have been localized to early endosomes, Rab24 is associated with both the ERlcis-Golgi region and late endosomes and is postulated to function in autophagy-related processes. Abbreviations: ER, endoplasmic reticulum; CGN, cis-Golgi network; TGN, trans-Golgi network; EE, early endosomes; LE, late endosomes. Arrows specify membrane traffic between compartments. Modified from: Simons and Zerial (1993). Protein sorting and vesicular traffic in the Golgi apparatus 113 nascent secretory vesicles from the TGN in endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors, and the effect can be antagonized by a stimulatory ARFI peptide (Austin and Shields, 1996).

Heterotrimeric G proteins Heterotrimeric G proteins are well known to be located on the plasma membrane and to be responsible for transmitting signals from seven transmembrane domain receptors to intracellular effectors such as ion channels, phospholipase c~l, and adenylate cyclase. More recently they have also been found on intracellular membranes, including Golgi membranes (Stow et al., 1991; Wilson et al., 1994; Stow, 1995; Denker et aI., 1996) and have been implicated in control of both exocytic and endocytic vesicular traffic. Initially and even now most of the evidence for a role for trimeric G proteins in intracellular trafficking is indirect and based on use of AIF4- or GTPys, pertussis toxin or cholera toxin, or agents such as mastoparan that mimic G protein effects. For instance, the first functional evidence for the involvement of Ga subunits in vesicular transport was the finding (Melancon et al., 1987) that AIF4-, which activates heterotrimeric but not small GTPases (Kahn, 1991), inhibits intra-Golgi transport in vitro. Similarly, Ga subunits have been implicated in ER to Golgi transport, budding of vesicles from the TGN destined for the apical and basolateral domains of the PM (Pimplikar and Simons, 1993), budding of regulated and constitutive vesicles from the TGN (Barr et aI., 1991) as well as formation of endocytic vesicles, fusion of incoming vesicles with endosomes (Colombo et aI., 1994), and transcytosis (Bomsel and Mostov, 1992). It has been known for some time that Gao and GUi3 are involved in the last steps of exocytosis, namely fusion of secretion granules with the plasma membrane in several regulated secretory cells (Ntimberg and Ahnert-Hilger, 1996). The only direct evidence for a role of G proteins in trafficking through the GA comes from the work of Stow et al. (Stow et aI., 1991) in which overexpression of Gai3 was found to slow transport of a secretory protein, a heparan sulfate proteoglycan, through the GA. Various theories have been put forth as to the specific role that G proteins play in traffic control. Donaldson et al. (1991) provided evidence that an AIF4--sensitive G protein is essential for assembly of coats on COP I-coated vesicles. de Almeida et al. (1993) have provided evidence that binding of p200 to Golgi membranes is regulated by heterotrimeric G proteins. Others have proposed that G proteins are required for vesicle budding (Barr et aI., 1992), vesicle fusion (in the case of secretion granules), or the efficiency of protein sorting (Bomsel and Mostov, 1992; Pimplikar and Simons, 1993). However, there is little direct evidence for any of these sugges• tions. Interestingly, heterotrimeric G proteins interact with ARF and may thereby modulate vesicle budding (Colombo et aI., 1995; Franco et al., 1995). 114 M.G. Farquhar and H.-P. Hauri

In summary, the specific role (or roles) of G proteins in the control of trafficking have not been elucidated. Moreover, nothing is known about the nature of the signal transduction cascades on intracellular membranes, and we do not know if the paradigm that applies to G protein signalling at the plasma membrane can be extrapolated to intracellular membranes (Helms, 1995). In contrast to the well-characterized receptor-G-protein-effector systems at the plasma membrane, no putative effectors or receptors have been identified on intracellular membranes. With the excep• tion of the KDEL receptor involved in retrieval of ER content proteins, no seven transmembrane domain receptors have been found so far on intracellular membranes. Clarification of the role of G proteins in control of vesicular traffic through the Golgi is sorely needed.

Phosphoinositides Various phosphoinositides can be generated from the membrane lipid phosphatidylinositol (PtIns) by different lipid kinases. The pathway by which the second messengers DAG and inosi• tol-l,4,5-trisphosphate are generated from PtIns-4,5-bisphosphate by phospholipase C is well known. Recently, it has become evident that phosphorylated Ptlns as well as soluble inositol polyphosphates (InsPPs) play essential roles in vesicular traffic, distinct from their role in classi• cal signalling pathways (De Camilli et aI., 1996). The highly charged phosphoinositides are thought to function at specific membrane sites by selectively recruiting or activating key proteins. The lipid kinases act in concert with specific phosphatases that either inactivate the inositide signalling pathway or convert the phosphoinositide into another isoform, that in turn triggers a downstream event. The first evidence that phosphoinositides may be involved in Golgi trafficking came from analysis of the yeast See14 gene that is required for post-Golgi secretory traffic (Novick et aI., 1980). See14 encodes a PtdIns transfer protein (Bankaitis et al., 1990) which is believed to serve as a sensor that maintains the appropriate phospholipid composition in the GA to allow vesicle budding from the TGN (McGee et aI., 1994; Skinner et aI., 1995; Alb et aI., 1996). Phosphoinositides are also strongly implicated in vesicular transport from the TGN to the vacu• ole (the yeast equivalent of lysosomes). This vesicular step requires the PtdIns3-kinase Vps34p. Localized production of PtdIns-3 phosphate by Vps34p may activate or recruit effector molecules such as coat proteins (Stack et aI., 1995; Seaman et aI., 1996). It is likely that phosphoinositides function at various steps of vesicular transport, for instance in budding. The downstream effector of ARFl, phospholipase D, is regulated by PtdIns(4,5)P2 (Terui et aI., 1994), and COP I binds InsPPs (Fleischer et aI., 1994). Similarly, clathrin adaptor complex AP2 binds InsPPs (Gaidarov et al., 1996). In some pathways vesicle fission may also be controled by phosphoinositides since dynamin is known to bind PtdIns(4,5)P2 and PtdIns (4,5,)3 via its PH domain (Zheng et aI., 1996; Urrutia et aI., 1997). An involvement in docking/fusion is Protein sorting and vesicular traffic in the Golgi apparatus 115 also likely as Rab5, which controls the fusion of endocytotic vesicles with endosomes, and homo• typic fusion of endosomes is regulated by PI-3 kinase (Li et al., 1995). A link to the cytoskeleton is illustrated by the fact that Rho GTPase binds and activates PtdIns(4)-5 kinase. The exact role played by phosphoinositides in the different steps of vesicular transport is not known, but a common theme appears to be regulation of protein recruitment. In that sense phos• phorylation and dephosphorylation of lipids or lipid-derived molecules is reminiscent of the phosphorylation and dephosphorylation of proteins. As with heterotrimeric G proteins, the sig• nalling cascades on intracellular membranes remain to be elucidated. An exciting avenue for future research is to elucidate how crosstalk between small GTPases, heterotrimeric G proteins and phosphoinositides coordinates vesicular traffic both at steady state and under conditions of signalling events that call for an adaptation of secretory activity.

Conclusions

The period covered in this review (1981-97) has seen a frenzy of research activity focused on the GA. As a result we have obtained a much deeper understanding of this organelle, the nature of its compartments, and control of traffic to and through it. In particular we have taken great strides toward an understanding of the molecular mechanisms involved in protein sorting and vesicular transport along the exocytic pathway. We now have a partial inventory of crucial proteins in• volved in vesicle budding and fission from the donor compartment and its recognition and fusion with its target. We also understand that signalling molecules - once believed to be restricted to the plasma membrane - are involv~d in traffic control. This period of rapid advance was facilitated by the convergence of information from such diverse sources as in vitro assays, biochemical analysis of Golgi fractions, immunocytochemical localization of proteins to pre-Golgi, Golgi and post• Golgi compartments, analysis of yeast mutants and characterization of synaptic vesicle proteins. Given the sophistication of our current understanding of the GA a student of the 1990s might be inclined to believe that this field is closed, and there is nothing much left to be done except fill in details. Nothing could be further from the truth. To begin with, we still have little understanding of the specific mechanisms involved in protein sorting at each step along the exocytic pathway. Moreover, few of the resident Golgi proteins have been purified and characterized. For example, there are estimated to be over 200 enzymes involved in glycosylation, many of which are assumed to be Golgi enzymes, and only a handful have been characterized. The mechanisms for retention of these and other Golgi resident proteins are also still poorly understood. Likewise, the contri• bution of cisternal maturation to anterograde protein transport through the GA remains to be elucidated. The biggest remaining challenge is to know how traffic is controlled - not only each 116 M.G. Farquhar and H.-P. Hauri vesicular transport step, but also how traffic control is integrated with other cellular needs. One of the important discoveries of this period is the demonstration that signalling molecules - small GTPases, heterotrimeric G proteins and phosphoinositides - once thought to be limited to the plasma membrane, are associated with intracellular compartments. This raises the question of whether the role of these signalling molecules is limited to regulation of vesicular transport as thought at present or whether, as seems likely, they are involved in signalling to other organelles including the nucleus where they might be expected to have a general influence on integration of cell behavior. These are only a few of the important avenues for investigation that lie ahead.

Acknowledgments The authors would like to thank Jeff Blenker for his patience and endurance in the preparation of this manuscript and Michael McCaffery for preparation of the figures. We thank Drs J. Stinchcombe and Colin Hopkins for providing Figure 4, Dr. Thomas Bachi for Figure 6, Dr. Lelio Orci for Figure 10, and Dr. Marino Zerial for Figure 14. We also thank the many people who provided preprints of their work prior to publication. Work in Marilyn Farquhar's laboratory was supported by grants from the National Institutes of Health (CA58689 and DKI7780), and work in Hans-Peter Hauri's laboratory was supported by the Swiss National Science Foundation, the Cantons of Basel, Sandoz Pharma Ltd. (Basel), and the Emilia Guggenheim-Schnurr Foundation.

References

Alb, J.G., Kearns, M.A. and Bankaitis, V.A. (1996) Phospholipid metabolism and membrane dynamics. Curro Opin. Cell BioI. 8: 534-541. Allan, D. and Kallen, K.J. (1993) Transport of lipids to the plasma membrane in animal cells. Prog. Lipid Res. 32: 195-219. Anderson, RG., Kamen, B.A., Rothberg, K.G. and Lacey, S.W. (1992) Potocytosis: sequestration and transport of small molecules by caveolae. Science 255: 410-411. Anderson, RG. and Pathak, RK. (1985) Vesicles and cisternae in the trans-Golgi apparatus of human fibroblasts are acidic compartments. Cell 40: 635-643. Andreis, P.G., Rebuffat, P., Belloni, A.S., Neri, G., Cavallini, L., GOllardo, G., Mazzocchi, G., Coi, A., Malendo• wicz, L.K. and Nussdorfer, G.G. (1989) Stereological and functional investigations on isolated adrenocortical cells: zona fasciculatalreticularis cells of chronically ACTH-treated rats. Cell Tiss. Res. 258: 43-51. Aniento, F., Feng, G., Parton, R.G. and Gruenberg, J. (1996) An endosomal b-COP is involved in the pH• dependent formation of transport vesicles destined for late endosomes. 1. Cell Bioi. 133: 29-41. Anthony, C., Cibert, C., Geraud, G., Santa-Maria, A., Maro, B., Mayau, V. and Goud, B. (1992) The small GTP• binding protein rab6p is distributed from medial-Golgi to the trans-Golgi network as determined by a confocal microscopy approach. J. Cell Sci. 103: 785-796. Aridor, M. and Balch, W.E. (1996) Principles of selective transport: coat complexes hold the key. Trends Cell Bioi. 6: 315-320. Aridor, M., Bannykh, S.I., Rowe, T. and Balch, W.E. (1995) Sequential coupling between COP II and COP I vesicle coats in endoplasmic reticulum to Golgi transport. 1. Cell BioI. 131: 875-893. Austin, C.D. and Shields, D. (1996) Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors. J. Cell BioI. 135: 1471-1483. Balch, W.E., McCaffery, J.M., Plutner, H. and Farquhar, M.G. (1994) Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell 76: 841-852. Banfield, D.K., Lewis, c., Rabouille, c., Warren, G. and Pelham, H.R (1994) Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains. J. Cell BioI. 127: 357-371. Bankaitis, V.A., Aitken, J.R, Cleves, A.E. and Dowhan, W. (1990) An essential role for a phospholipid transfer protein in yeast Golgi function. Nature 347: 561-562. Bannykh, S.1. and Balch, W.E. (1997) Membrane dynamics at the endoplasmic reticulum - Golgi interface. J. Cell BioI.; in press. Protein sorting and vesicular traffic in the Golgi apparatus 117

Bannykh, S.l., Rowe, T. and Balch, W.E. (1996) The organization of endoplasmic reticulum export complexes. J. Cell BioI. 135: 19-35. Barlowe, c., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.P., Ravazzola, M., Arnherdt, M. and Schekrnan, R (1994) COP II: A membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77: 895-907. Barr, F.A., Leyte, A. and Huttner, W.B. (1992) Trimeric G proteins and vesicle formation. Trends Cell Bioi. 2: 91-94. Barr, P.A., Leyte, A., Mollner, S., Pfeuffer, T., Tooze,S.A. and Huttner, W.B. (1991) Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules. FEBS Lett. 294: 239-243. Bartles, J.R, Feracci, H.M., Stieger, B. and Hubbard, A.L. (1987) Biogenesis of the rat hepatocyte plasma membrane in vivo: Comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. J. Cell Bioi. 1987: 1241-1251. Becker, B., Bolinger, B. and Melkonian, M. (1995) Anterograde transport of algal scaler through the Golgi complex is not mediated by vesicles. Trends Cell BioI. 5: 305 - 307. Beckers, CJ.M., Keller, D.S. and Balch, W.E. (1987) Semi-intact cells permeable to macromolecules: use in reconstitution ofprotein transport from the endoplasmic reticulum to the Golgi complex. Cell 50: 523-534. Bednarek, S.Y., Orci, L. and Schekrnan, R. (1996) Traffic COPs and the formation of vesicle coats. Trends Cell BioI. 6: 468-473. Bednarek, S.Y., Ravazzola, M., Hosobuchi, M., Amherdt, M., Perrelet, A., Schekrnan, Rand Orci, L. (1995) COP 1- and COP II-coated vesicles bud directly from the endoplasmic reticulum. Cell 83: 1183-1196. Belden, W.J. and Barlowe, C. (1996) Erv25p, a component of COP II-coated vesicles, forms a complex wilth Emp24p that is required for efficient endoplasmic reticulum to Golgi transport. J. BioI. Chem. 271: 26939• 26946. Bennett, M.K. (1995) SNAREs and the specificity of transport vesicle targeting. Curro Opin. Cell BioI. 7: 581• 586. Bennett, M.K. and Scheller, R.H. (1993) The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl Acad. Sci. USA 90: 2559-2563. Bennett, M.K. and Scheller, R.H. (1994) A molecular description of synaptic vesicle membrane trafficking. Ann. Rev. Biochem. 63: 63-100. Bergeron, J.J.M., Brenner, M.B., Thomas, D.Y. and Williams, D.B. (1994) Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Cell BioI. 19: 124-128. Boll, W., Ohno, H., Songyang, Z., Rapoport, 1., Cantley, L.C., Bonifacino, J.S. and Kirchhausen, T. (1996) Sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin AP-2 complexes. EMBO J. 15: 5789-5795. Bomsel, M. and Mostov, K. (1992) Role of heterotrimeric G proteins in membrane traffic. Molec. BioI. Cell 3: 1317-1328. Bonifacino, J.S. (1996) Reversal of fortune for nascent proteins. Nature 384: 405-406. Bosshard, H., Humphrey, J., Deignan, E., Davidson, J., Drazba, J., Yuan, L., Oorschot, V., Peters, PJ. and Bonifacino, J.S. (1994) The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomalllysosomal system. J. Cell BioI. 126: 1157-1172. Bretscher, M.S. and Munro, S. (1993) Cholesterol and the Golgi apparatus. Science 261: 1280-1281. Brown, RM. Jr and Willison, J.H.M. (1977) In: B.R. Brinkley and K.R. Porter (eds): International Cell Biology, 1976-1977, The Rockefeller University Press, New York, pp 267-283. Burgess, T.L. and Kelly, RB. (1987) Constitutive and regulated secretion of proteins. Ann. Rev. Cell BioI. 3: 243-293. Burke, J., Pettitt, J.M., Schachter, H., Sarkar, M. and Gleeson, P.A. (1992) The transmembrane and flanking sequences of ~1,2-N-acetylglucosaminyltransferaseI specify medial-Golgi localization. J. BioI. Chem. 267: 24433-24440. Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C.L. and Chabre, M. (1996) A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains. Nature 384: 481-484. Chavez, R.A., Miller, S.G. and Moore, H.P. (1996) A biosynthetic regulated secretory pathway in constitutive secretory cells. J. Cell BioI. 133: 1177-1191. Chavrier, P., Parton, RG., Hauri, H.-P., Simons, K. and Zerial, M. (1990) Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell 62: 317-329. Chin, OJ., Luskey, K.L., Anderson, RG.W., Faust, J.R., Goldstein, J.L. and Brown, MJ. (1982) Appearance of crystalloid endoplasmic reticulum in compactin resistant Chinese hamster cells with a 500-fold increase in 3• hydroxy-3-methylglutaryl-coenzyme A reductase. Proc. Natl Acad. Sci. USA 70: 1185-1189. Clermont, Y., Xia, L. Rambourg, A., Turner, J.D. and Hermo, L. (1993) Transport of casein submicellles and formation of secretion granules in the Golgi apparatus of epithelial cells of the lactating mammary gland of the rat. Anatomical Record 235: 363-373. Cole, N.B., Smith, C.L., Sciaky, N., Terasaki, M., Edidin, M. and Lippincott-Schwartz, J. (1996) Diffusional mobility of Golgi proteins in membranes of living cells. Science 273: 797- 80 1. 118 M.G. Farquhar and H.-P. Hauri

CoIley, K.J. (1977) Golgi localization of glycosyltransferases - more questions than answers. Glycobiology 7: l• B. Colombo, M.I., Inglese, J., D'Souza-Schorey, C., Beron, W. and Stahl, P.D. (1995) Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic. J. Bioi. Chern. 270: 24564-2457l. Colombo, M.I., Mayorga, L.S., Nishimoto, I., Ross, E.M. and Stahl, P.D. (1994) Gs regulation of endosome fusion suggests a role for signal transduction pathways in endocytosis. J. BioI. Chern. 269: 14919-14923. Colomer, V., Kicska, G.A. and Rindler, M.J. (1996) Secretory granule content proteins and the luminal domains of granule membrane proteins aggregate in vitro at mildly acidic pH. J. BioI. Chern. 271: 48-55. Conibear, E. and Stevens, TH. (1995) Vacuolar biogenesis in yeast: Sorting out the sorting proteins. Cell 83: 513-516. Conrad, P.A., Smart, E.J., Ying, Y.S., Anderson, R.G. and Bloom, G.S. (1995) Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps. J. Cell BioI. 131: 1421-1433. Cool, D.R., Fenger, M., Snell, C.R. and Loh, Y.P. (1995) Identification of the sorting signal motif within proopiomelanocortin for the regulated secretory pathway. J. BioI. Chern. 270: 8723-8729. . Cool, D.R., Normant, E., Shen, F., Chen, H.-C., PanneIl, L., Zhang, Y. and Loh, Y.P. (1997) Carboxypeptidase E is a regulated secretory pathway sorting receptor: Genetic obliteration leads to endocrine disorders in Cpefat mice. Cell 88: 73 - 83. Coorssen, J.R, Schmitt, H. and Almers, W. (1996) Ca2+ triggers massive exocytosis in Chinese hamster ovary ceIls.EMBOJ.15:3787-379l. Cosson, P., DemoIliere, C., Hennecke, S., Duden, R. and Letourneur, F. (1996) Delta- and zeta-COP, two coatomer subunits homologous to c1athrin-associated proteins, are involved in ER retrieval. EMBO J. 15: 1792-1798. Cosson, P. and Letourneur, F. (1994) Coatomer interaction with di-Iysine endoplasmic reticulum retention motifs. Science 263: 1629-163l. Dahan, S., Ahluwalia, J.P., Wong, L., Posner, B.1. and Bergeron, J.1. (1994) Concentration of intraceIlular hepatic apoliprotein E in Golgi apparatus saccular distensions and endosomes. J. Cell BioI. 127: 1859-1869. Dascher, C., Matteson, J. and Balch, W.E. (1994) Syntaxin 5 regulates endoplasmic reticulum to Golgi transport. J. Bioi. Chern. 269: 29363-29366. de Almeida, J.B., Doherty, J., AusieIlo, D.A. and Stow, J.L. (1993) Binding of the cytosolic p200 protein to Golgi membranes is regulated by heterotrimeric G proteins. J. Cell Sci. 106(4): 1239-1248. De CamiIli, P., Ernr, S.D., McPherson, P.S. and Novick, P. (1996) Phosphoinositides as regulators in membrane traffic. Science 271: 1533 -1538. De Camilli, P. and Takei, K. (1996) Molecular mechanisms in synaptic vesicle endocytosis and recycling. Neuron 16: 481-486. DeIl'Angelica, E.C., Ohno, H., Ooi, C.E., Rabinovich, E., Roche, K.W. and Bonifacino, J.S. (1997) AP-3: an adaptor-like protein complex with ubiquitous expression. EMBO J. 16: 917-928. Denker, S., McCaffery, J.M., Palade, G.E., Insel, P.A. and Farquhar, M.G. (1996) Differential distribution of a subunits and 13Y subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas. J. Cell Bioi. 133: 1027 -1040. Dirac-Svejstrup, A.B., Soldati, T., Shapiro, A.D. and Pfeffer, S.R. (1994) Rab-GDI present functional Rab9 to the intraceIlular transport machinery and contributes selectively to Rab9 membrane recruitment. J. BioI. Chern. 269: 15427-15430. Donaldson, J.G., Finazzi, D. and Klausner, R.D. (1992) Brefeldin A inhibits Golgi-membrane-catalyzed exchange of guanine nucleotide onto ARF protein. Nature 360: 350-352. Donaldson, J.G., Kahn, RA., Lippincott-Schwartz, J. and Klausner, R.D. (1991) Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science 254: 1197-1199. Donaldson, J.G. and Klausner, RD. (1994) ARF: a key regulatory switch in membrane traffic and organeIle structure. Curro Opin. Cell BioI. 6: 527-532. Draper, RK., Goda, Y., Brodsky, F.M. and Pfeffer, S.R. (1990) Antibodies to c1athrin inhibit endocytosis but not recycling to the trans-Golgi network in vitro. Science 248: 1539-154l. Duden, R., AIIan, V. and Kreis, T. (l991a) Involvement of 13-COP in membrane traffic through the Golgi complex. Trends Cell BioI. 1: 14-19. Duden, R, Griffiths, G., Frank, R., Argos, P. and Kreis, TE. (l99Ib) 13-COP, a 110 kD protein assciated with non-c1athrin-coated vesicles and the Golgi complex, shows homology to 13-adaptin. Cell 64: 649-665. Dunphy, W.G., Brands, R. and Rothman, J.E. (1985) Attachment of terminal N-acetylglucosamine to asparagine• linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell 40: 463-472. Dunphy, W.G. and Rothman, J.E. (1983) Compartmentation of asparagine-linked oligosaccharide processing in the Golgi appartus. J. Cell BioI. 97: 270-275. Dupree, P., Parton, R.G., Raposo, G., Kurzchalia, TV. and Simons, K. (1993) Caveolae and sorting in the trans• Golgi network of epithelial ceIls. EMBO J. 12: 1597-1605. Dustin, M.L., Baranski, TJ., Sampath, D. and Kornfeld, S. (1995) A novel mutagenesis strategy identifies distantly spaced amino acid sequences that are required for the phosphorylation of both the oligosaccharides of procathepsin D by N-acetylglucosamine I-phosphotransferase. J. Bioi. Chern. 270: 170-179. Protein sorting and vesicular traffic in the Golgi apparatus 119

Farquhar, M.G. (1985) Progress in unraveling pathways of Golgi traffic. Ann. Rev. Cell BioI. I: 447-488. Farquhar, M.G. (1991) Protein traffic through the Golgi. In: C. Steer and J. Hanover (eds): Intracellular Trafficking ofProteins, Cambridge University Press, Cambridge, pp 431-471. Farquhar, M.G. and Palade, G.E. (1981) The Golgi apparatus (complex}-(1954-1981}-from artefact to center stage. J. Cell BioI. 91: 77S-103S. Faulstich, D., Auerbach, S., Orci, L., Ravazzola, M., Wegchlingel, S., Lottspeich, F., Stenbeck, G., harter, C., Wieland, F.T. and Tschochner, H. (1996) Architecture of coatomer: molecular characterization ofdelta-COP and protein interactions within the complex. J. Cell BioI. 135: 53-61. Ferro-Novick, S. and Jahn, R (1994) Vesicle fusion from yeast to man. Nature 370: 191-193. Ferro-Novick, S. and Novick, P. (1993) The role of GTP-binding proteins in transport along the exocytic path• way. Ann. Rev. Cell BioI. 9: 575-599. Fiedler, K., Kobayashi, T., Kurzchalia, T.V. and Simons, K. (1993) Glycosphingolipid-enriched, detergent• insoluble complexes in protein sorting in epithelial cells. Biochemistry 32: 6365-6375. Fiedler, K., Lafont, F., Parton, R.G. and Simons, K. (1995) Annexin XIllb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. J. Cell BioI. 128: 1043-1053. Fiedler, K., Parton, R.G., Kellner, R., Etzold, T. and Simons, K. (1994) VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells. EMBO J. 13: 1729-1740. Fiedler, K. and Simons, K. (1995) The role ofN-glycans in the secretory pathway. Cell 81 : 309-312. Fiedler, K., Veit, M., Stamnes, M.A. and Rothman, J.E. (1996) Biomodal interaction of coatomer with the p24 family of putative cargo receptors. Science 273: 1396-1399. Fleischer, B., Xie, J., MayrIeitner, M., Shears, S.B., Palmer, DJ. and Fleischer, S. (1994) Golgi coatomer binds, and forms K(+)-selective channels gated by, inositol polyphosphates. J. BioI. Chem. 269: 17862-17832. Franco, M., Paris, S. and Chabre, M. (1995) The small G-protein ARFIGDP binds to the Gt beta gamma subunit of transducin, but not to Gt alpha GDP-Gt beta gamma. FEBS Lett. 362: 286-290. Frohman, M.A. and Morris, AJ. (1996) Rho is only ARF the story. Phospholipid signalling. Curro BioI. 6: 945• 947. Fujimoto, T. (1993) Calcium pump of the plasma membrane is localized in caveolae. J. Cell BioI. 120: 1147• 1157. Futter, C.E., Connolly, C.N., Cutler, D.F. and Hopkins, C.R. (1995) Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface. J. BioI. Chem. 270: 10999-11003. Gaidarov, I., Chen, Q., Falck, J.R, Reddy, K.K. and Keen, J.H. (1996) A functional phosphatidylinositol 3,4,5• trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for the endocytic pathway. J. Bioi. Chem. 271: 20922-20929. Galteau, M.M., Antoine, B. and Reggio, H. (1985) Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver. EMBO J. 4: 2793-2800. Gaynor, E.C., te Heesen, S., Graham, T.R, Aebi, M. and Ernr, S.D. (1994) Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Bioi. 127: 653-665. Gething, M.-J. and Sambrook, J. (1992) Protein folding in the cell. Nature 355: 33-45. Geuze, HJ., Slot, J.W., Strous, GJ., Hasilik, A. and Von Figura, K. (1984) Ultrastructural localization of the mannose 6-phosphate receptor in rat liver. J. Cell BioI. 98: 2047-2054. Ghitescu, L., Fixman, A., Simionescu, M. and Simionescu, N. (1986) Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis. J. Cell BioI. 102: 1304-1311. Ghosh, S., Strum, J.C., Sciorra, V.A., Daniel, L. and Bell, RM. (1996) Raf-I kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-I in 12-0-tetradecanoylphorbol-l3-acetate-stimulated Madin-Darby canine kidney cells. J. BioI. Chem. 271: 8472• 8480. Gimeno, RE., Espenshade, P. and Kaiser, C.A. (1995) SED4 encodes a yeast endoplasmic reticulum protein that binds Secl6p and participates in vesicle formation. J. Cell BioI. 131: 325-338. Glickman, J., Croen, K., Kelly, S. and AI-Awqati, Q. (1983) Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance. J. Cell Bioi. 97: 1303-1308. Glickman, J.N., Conibear, E. and Pearse, B.M.F. (1989) Specificity of binding of clathrin adaptors to signals on the mannose 6-phosphatelinsulin-like growth factor II receptor. EMBO J. 8: 1041-1047. Goldberg, D.E. and Kornfeld, S. (1983) EVidence for extensive subcellular oganization and asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation. J. BioI. Chem. 258: 3159-3165. Gonatas, J.O., Mezitis, S.G., Stieber, A., Fleischer, B. and Gonatas, N.K. (1989) MG-160. A novel sialoglycoprotein of the medial cisternae of the Golgi apparatus (published erratum appears in J. BioI. Chem. 1989,264: 4264). J. BioI. Chem. 264: 646-653. Goud, B., Zahraoui, A., Tavitian, A. and Saraste, J. (1990) Small GTP-binding protein associated with the Golgi cisternae. Nature 345: 553-556. Grasse, P.P. (1957) C.R. Acad. Sci. (Paris) 245: 1278-1281. Griffiths, G., Ericsson, M., Krijnse-Locker, J., Nilsson, T., Goud, B., Soling, H.D., Tang, B.L., Wong, S.H. and Hong, W. (1994) Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells. J. Cell BioI. 127: 1557-1574. 120 M.G. Farquhar and H.-P. Hauri

Griffiths, G., Hoflack, B., Simons, K., Mellman, 1. and Kornfeld, S. (1988) The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell 52: 328-341. Griffiths, G. and Simons, K. (1986) The trans-Golgi network: sorting at the exit site of the Golgi complex. Science 234: 438-443. Griffiths, G., Warren, G., Quinn, P., Mathieu-Costello, O. and Hoppeler, H. (1984) Density of newly synthesized plasma membrane proteins in intracellular membranes. 1. Stereological studies. J. Cell BioI. 98: 2133-2141. Hammond, S.M., Jenco, J.M., Nakashima, S., Cadwallader, K., Gu, Q., Cook, S., Nozawa, Y., Prestwich, G.D., Frohman, M.A. and Morris, A.J. (1997) Characterization of two alternately spliced forms of phospholipase 01. Activation of the purified enzymes by phosphatidylinositol4,5-bisphosphate, ADP-ribosylation factor, and RHO family monomeric GTP-binding proteins and protein kinase C. J. BioI. Chen/. 272: 3860-3868. Hara-Kuge, S., Kuge, 0., Orci, L., Amherdt, M., Ravazzola, M., Wieland, F.T. and Rothman, J.E. (1994) En block incorporation of coatomer subunits during the assembly of COP-coated vesicles. J. Cell BioI. 124: 883-893. Harter, C., Pavel, J., Coccia, F., Draken, E., Wegehingel, S., Tschochner, H. and Wieland, F. (1996) Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic di-Iysine motif ofmembrane proteins of the early secretory pathway. Proc. Natl Acad.. Sci. USA 93: 1902-1906. Harter, C. and Wieland, F. (1995) The secretory pathway: mechanisms of protein sorting and transport. Biochim. Biophys. Acta 286: 75-93. Hauri, H.-P. and Schweizer, A (1992) The endoplasmic reticulum-Golgi intermediate compartment. Curro Opin. Cell BioI. 4: 600-608. Hauri, H.-P. and Schweizer, A (1997) The ER - Golgi membrane system: Compartmental organization and protein traffic. In: J.F. Hoffmann and J.D. Jamieson (eds): Handbook of Physiology. Oxford University Press for the American Physiological Society, New York, pp 605-642. Hayes, B.K., Freeze, H.H. and Varki, A (1993) Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labelled by UDP-[6-3H]N-acetylglucosamine. J. Bioi. Chem. 268: 16139-16154. Hayes, B.K. and Varki, A. (1993) Biosynthesis of oligosaccharides in intact Goigi preparations from rat liver. Analysis of N-linked glycans labelled by UDP-[6-3H]galactose, CMP-[9-3H]N-acetylneuraminic acid, and [acetyl-3H]acetyl-coenzyme A J. BioI. Chem. 268: 16155-16169. Helenius, A. (1994) How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Malec. BioI. Cell 5: 253-265. Helenius, A, Marquardt, T. and Braakrnan, 1. (1992) The endoplasmic reticulum as a protein-folding compartment. Trends Cell BioI. 2: 227-231. Helms, J.B. (1995) Role of heterotrimeric GTP binding proteins in vesicular protein transport: indications for both classical and alternative G protein cycles. FEBS Lett. 369: 84-88. Helms, J.B. and Rothman, J.E. (1992) Inhibition by brefeldin A of a Golgi membrane enzyme that catalyzes exchange of guanine nucleotide bound to ARF. Nature 360: 352-354. Hinshaw, J.E. and Schmid, S.L. (1995) Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 374: 190-192. Hobman, T.e., Woodward, L. and Farquhar, M.G. (1992) The rubella virus EI glycoprotein is arrested in a novel, post-ER pre-Golgi compartment. J. Cell BioI. 118: 795-812. Holstein, S.E., Ungewickell, H. and Ungewickell, E. (1996) Mechanism of c1athrin basket dissociation:separate functions of protein domains of the DnaJ homologue auxilin. J. Cell Bioi. 135:. Hong, W. (1996) Protein Trafficking along the Exocytotic Pathway. R.G. Landes Company, Austin. Humphrey, J.S., Peters, P.J., Yuan, L.C. and Bonifacino, J.S. (1993) Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing motif. J. Cell BioI. 120: 1123-1135. Hunziker, W. and Geuze, H.J. (1996) Intracellular trafficking of lysosomal membrane proteins. Bioessays 18: 379• 389. Hurtley, S.M. and Helenius, A (1989) Protein oligomerization in the endoplasmic reticulum. Ann. Rev. Cell BioI. 5: 277-307. Huttner, W.B. (1988) Tyrosine sulfation and the secretory pathway. Ann. Rev. Physiol. 50: 363-376. Huttner, W.B., Gerdes, H.-H. and Rosa, P. (1991) The granin (chromogranin/secretogranin) family. Trends BioI. Sci. 16: 27-30. Ikonen, E., Tagaya, M., Ullrich, 0., Montecucco, C. and Simons, K. (1995) Different requirements of NSF, SNAP, and Rab proteins in apical and basolateral transport in MOCK cells. Cell 81: 571-580. Itin, C., Foguet, M., Kappeler, F., K1umperman, J. and Hauri, H.-P. (I995a). Recycling of the endoplasmic reticulurn/Golgi intermediate compartment protein ERGIC-53 in the secretory pathway. Biochem. Soc. Trans. 23: 541-544. Itin, C., Roche, A-e., Monsigny, M. and Hauri, H.-P. (1996) ERGlC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins. Malec. BioI. Cell 7: 483-493. ltin, C., Schindler, R. and Hauri, H.-P. (1995b). Targeting of protein ERGlC-53 to the ERlERGlC/cis-Golgi recycling pathway. J. Cell BioI. 131: 57-67. Jackson, M.R., Nilsson, T. and Peterson, P.A. (1990) Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J. 9: 3153-3162. Protein sorting and vesicular traffic in the Golgi apparatus 121

Jackson, M.R., Nilsson, T. and Peterson, P.A. (1993) Retrieval of transmembrane proteins to the endoplasmic reticulum. J. Cell BioI. 121: 317-333. Jin, M., Saucan, L., Farquhar, M.G. and Palade, G.E. (1996) Rabla and multiple other Rab proteins are associated with the transcytotic pathway in rat liver. J. BioI. Chem. 271: 30105-30113. Johnson, K.F. and Kornfeld, S. (1992) A his-leu-leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. J. BioI. Chem. 267: 17110-17115. Jones, S.M., Crosby, J.R., Salamero, J. and Howell, K.E. (1993) A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network. J. Cell Bioi. 122: 775-788. Jung, L.J., Kreiner, T. and Scheller, R.H. (1993) Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting. J. Cell Bioi. 121: 11-21. Kahn, R.A. (1991) Fluoride is not an activator of the smaller (20-25 kD) GTP-binding proteins. J. BioI. Chem. 266: 15595-15597. Kain, R., Angata, K., Kerjaschki, D. and Fukuda, M. (1996) Molecular cloning and expression of human trans• Golgi network 38 glycoprotein that contains unique extracellular sequence. Molec. BioI. Cell 7: 441a. Kasper, D., Dittmer, F., von Figura, K. and Pohlmann, R. (1996) Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes. J. Cell Bioi. 134: 615-623. Kelly, R.B. (1985) Pathways of protein secretion in eukaryotes. Science 230: 25-32. Kelly, R.B. (1990) Microtubules, membrane traffic, and cell organization. Cell 61: 5-7. Kelly, R.B. (1991) Secretory granule and synaptic vesicle formation. Curro Opin. Cell BioI. 3: 654-660. Kelly, R.B. (1993) Storage and release of neurotransmitters. Cell 72: 43-53. Kirchhausen, T. (1993) Coated pits and coated vesicles - sorting it all out. Curro Struct. BioI. 3: 182-188. Klausner, R.D., Donaldson, J.G. and Lippincott-Schwartz, J. (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Bioi. 116: 1071-1080. Klein, J., Chalifa, V., Liscovitch, M. and Loffelholz, K. (1995) Role of phospholipase D activation in nervous system physiology and pathophysiology. J. Neurochem. 65: 1445-1455. Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked oligosaccharides. Ann. Rev. Biochem. 54: 631-664. Kornfeld, S. (1992) Structure and function of the mannose 6-phosphatelinsulinlike growth factor 11 receptors. Ann. Rev. Biochem. 61: 307-330. Kornfeld, S. and Mellman, I. (1989) The biogenesis of Iysosomes. Ann. Rev. Cell Bioi. 5: 483 -525. Kreis, T.E., Lowe, M. and Pepperkok, R. (1995) COPs regulating membrane traffic. Ann. Rev. Cell Bioi. 11: 677• 706. Kreis, T.E. and Pepperkok, R. (1994) Coat proteins in intracellular membrane transport. Curro Opin. Cell BioI. 6: 533-537. Ktistakis, N.T., Brown, H.A., Sternweis, P.C. and Roth, M.G. (1995) Phospholipase D is present on Golgi• enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A. Proc. Natl Acad. Sci. USA 92: 4952-4956. Ktistakis, N.T., Brown, H.A., Waters, M.G., Sternweis, P.C. and Roth, M.G. (1996) Evidence that phospholipase D mediates ADP ribosylation factor - dependent formation of Golgi coated vesicles. J. Cell BioI. 134: 295• 306. Ktistakis, N.T. and Roth, M.G. (1996) Protein sorting during endocytosis. In: S.M. Hurtley (ed.): Protein Targeting, IRL Press at Oxford University Press, Oxford, pp 176-212. Kurzchalia, T.V., Dupree, P., Parton, R.G., Kellner, R., Virta, H., Lehnert, M. and Simons, K. (1992) VIP21, a 21• kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. 1. Cell BioI. 118: 1003-1014. Kurzchalia, TV. and Parton, R.G. (1996) And still they are moving...: Dynamic properties of caveolae. FEBS Lett. 389: 52-54. Ladinsky, M.S., Kremer, J.R., Furcinitti, P.S., McIntosh, J.R. and Howell, K.E. (1994) HVEM tomography of the trans-Golgi network: structural insights and identification of lace-like vesicle coat. J. Cell Bioi. 127: 29-38. Lahtinen, U., Hellman, U., Wernstedt, c., Saraste, J. and Pettersson, R.F. (1996) Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein. J. BioI. Chem. 271: 4031-4037. Lamaze, C. and Schmid, S.L. (1995) The emergence of c1athrin-independent pinocytic pathways. Curro Opin. Cell BioI. 7: 573-580. Lambert, M., Bui, N.-D. and Christophe, J. (1990) Novel GTP-binding proteins in plasma membranes and zymogen granule membranes from rat pancreas and in pancreatic AR 4-2J cell membranes. FEBS Lett. 271: 19• 22. Leblond, C.P. (1989) Synthesis and secretion of collagen by cells of connective tissue, bone, and dentin. Anatomical Record 224: 123-138. Le Borgone, R., Griffiths, G. and Hoflack, B. (1996) Mannose 6-phosphate receptors and ADP-ribosylation factors cooperate for high affinity interaction of AP-l Golgi assembly proteins with membranbes. J. Bioi. Chem. 271: 2162-2170. 122 M.G. Farquhar and H.-P. Hauri

Le Borgone, R. and Hoflack, B. (1997) Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TNG. J. Cell BioI. 137: 335-345. Le Gall, A.H., Yeaman, C., Muesch, A and Rodriguez-Boulan, E. (1995) Epithelial cell polarity: New perspec• tives. Seminars Nephrol. 15: 272-284. Leitinger, B., Hille-Rehfeld, A. and Spiess, M. (1995) Biosynthetic transport of the asialoglycoprotein receptor HI to the cell surface occurs via endosomes. Proc. Natl Acad. Sci. USA 92: 10109-10113. Lepage-Lezin, A, Joseph-Bravo, P., Devilliers, G., Benedetti, L., Launay, J.M., Gomez, S. and Cohen, P. (1991) Prosomatostatin is processed in the Golgi apparatus of rat neural cells. J. BioI. Chem. 266: 1679-1688. Letoumeur, F., Gaynor, E.C., Hennecke, S., Demolliere, C., Duden, R., Emr, S.D., Riezman, H. and Cosson, P. (1994) Coatomer is essential for retrieval of di-Iysine-tagged proteins to the ER. Cell 79: 1199-1207. Lewis, MJ. and Pelham, H.R.B. (1996) SNARE-mediated retrograde traffic from the Golgi complex to the endoplasmic reticulum. Cell 85: 205-215. Li, G., D'Souza-Schorey, C., Barbieri, M.A., Roberts, R.L., Klippel, A, Williams, L.T. and Stahl, P.D. (1995) Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc. Natl Acad. Sci. USA 92: 10207-10211. Lindstedt, AD. and Hauri, H.-P. (1993) Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kD. Malec. Cell Bioi. 4: 679-693. Lindstedt, A.D. and Hauri, H.-P. (1997) Sequence and overexpression of GPP130/GIMPc: Evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein. Malec. BioI. Cell; in press. Lisanti, M.P., Scherer, P.E., Tang, Z. and Sargiacomo, M. (1994) Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Bioi. 4: 231-235. Liscovitch, M. and Cantley, L.C. (1995) Signal transduction and membrane traffic: The PITP/Phosphoinositide connection. Cell 81: 659-662. Liu, S.H., Wong, M.L., Craik, C.S. and Brodsky, F.M. (1995) Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs. Cell 83: 257-267. Lombardi, D., Soldati, T., Riederer, M.A, Goda, Y., Zerial, M. and Pfeffer, S.R. (1993) Rab9 functions in transport between late endosomes and the trans-Golgi network. EMBO J. 12: 677-682. Lotti, L.V., Torrisi, M.R., ETTa, M.C. and Bonatti, S. (1996) Morphological analysis of the transfer of VSV ts-045 G glycoprotein from the endoplasmic reticulum to the intermediate compartment in vero cells. Exp. Cell Res. 227: 323-331. Louvard, D., Kedinger, M. and Hauri, H.-P. (1992) The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures. Ann. Rev. Cell BioI. 8: 157 -195. Lowe, M. and Kreis, T.E. (1995) In vitro assembly and disassembly of coatomer. J. Bioi. Chem. 270: 31364• 31371. Lutcke, A., Parton, R.G., Murphy, C., Olkkonen, V.M., Dupree, P., Valencia, A, Simons, K and Zerial, M. (1994) Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression. J. Cell Sci. 107: 3437-48. Luzio, J.-P., Brake, 8., Bantin, G., Howell, K.E., Braghetta, P. and Stanley, KK (1990) Identification, sequencing, and expression of an integral membrane protein of the trans-Golgi network, TGN 38. Biochemistry J 270: 97• 102. Machamer, C.E. (1993) Targeting and retention of Golgi membrane proteins. Curro Opin. Cell BioI. 5: 606-612. Machamer, C.E. (1996) ER-Golgi membrane traffic and protein targeting. In: S. M. Hurtley (ed.): Protein Targeting, IRL Press at Oxford University Press, Oxford, pp 123-151. Machamer, C.E., Mentone, S.A., Rose, J.K and Farquhar, M.G. (1990) The EI glycoprotein of an avian coronavirus is targeted to the cis-Golgi complex. Proc. Natl Acad. Sci. USA 87: 6944-6948. Machamer, C.E. and Rose, J.K (1987) A specific transmembrane domain of a coronavirus El glycoprotein is required for its retention in the Golgi region. J. Cell Bioi. 105: 1205-1214. Mahaffey, D.T., Peeler, J.S., Brodsky, F.M. and Anderson, R.G. (1990) Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity. J. Bioi. Chem. 265: 16514-16520. Mandon, E., Kempner, E.S., Ishihara, M. and Hirschberg, C.B. (1994) A monomeric protein in the Golgi membrane catalyzes both N-deacetylation and N-sulfation of heparan sulfate. J. BioI. Chem. 269: 11729-11733. Marks, M.S., Ohno, H., Kirchhausen, T. and Bonifacino, S.J. (1997) Protein sorting by tyrosine-based signals: Adapting to the Ys and wherefores. Trends Cell BioI. 7: 124-128. Marks, M.S., Woodruff, L., Ohno, H. and Bonifacino, J.S. (1996) Protein targeting by tyrosine- and di-leucine• bases signals: Evidence for distinct saturable components. J. Cell BioI. 135: 341-354. Marti, U., Hauri, H.P. and Meyer, U.A. (1990) Induction of cytochrome P450 by phenobarbital in rat liver visualized by monoclonal antibody immunoelectron microscopy in situ. Eur. J. Cell BioI. 52: 193-200. Martinez, 0., Schmidt, A., Salamero, J., Hoflack, B., Roa, M. and Goud, B. (1994) The small GTP-binding protein rab6 functions in intra-Golgi transport. J. Cell BioI. 127: 1575-1588. Matter, K. and Mellman, 1. (1994) Mechanism of cell polarity: sorting and transport in epithelial cells. Curro Opin. Cell Bioi. 6: 545-554. Mayer, A., Wickner, W. and Haas, A. (1996) Secl8p (NSF)-driven release of Sec17p(alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85: 83 -94. Protein sorting and vesicular traffic in the Golgi apparatus 123

McGee, T.P., Skinner, H.B., Whitters, E.A., Henry, S.A. and Bankaitis, V.A. (1994) A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes. J. Cell BioI. 124: 273 -287. Melancon, P., Glick, B.S., Malhotra, V., Weidman, P.J., Serafini, T., Gleason, M., Orci, L. and Rothman, J.E. (1987) Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell 51: 1053 -1062. Mellman, I. (1996) Endocytosis and molecular sorting. Ann. Rev. Cell Develop. BioI. 12: 575-625. Mellman, I. and Simons, K. (1992) The Golgi complex: In vitro veritas? Cell 68: 829-840. Mendis-Handagama, S.M., Zirkin, B.R and Ewing, L.L. (1989) Comparison of components of the testis interstiti• um with testosterone secretion in hamster, rat, and guinea pig testes perfused in vitro. Am. J. Anat. 181: 12-22. Merisko, E.M., Fletcher, M. and Palade, G.E. (1986) The reorganization of the Golgi complex in anoxic pancreatic acinar cells. Pancreas 1: 95 -109. Miller, S.G. and Moore, H.-P. (1990) Regulated secretion. Curro Opin. Cell BioI. 2: 642-647. Mizuno, M. and Singer, SJ. (1993) A soluble secretory protein is first concentrated in the endoplasmic reticulum before transfer to the Golgi apparatus. Proc. Natl Acad. Sci. USA 90: 5732-5736. Montesano, R, Roth, J., Robert, A. and Orci, L. (1982) Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature 296: 651-653. Moore, H.-P.H., Orci, L. and Oster, G.F. (1988) Biogenesis of secretory vesicles. In: RC. Das and P.W. Robbins (eds): Protein Transfer and Organelle Biogenesis, Academic Press, San Diego, pp 521-561. Morinaga, N., Tsai, S.-C., Moss, J. and Vaughan, M. (1996) Isolation of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP ribosylation factor (ARF)1 and ARF3 that contains a Sec7-like domain. Proc. Natl Acad. Sci. USA 93: 12856-12860. MoITt~, D.J., Franke, W.W., Deumling, B., Nyquist, S.E. and Outracht, L. (1971) Golgi apparatus function in membrane flow and differentiation: Origin of plasma membranes from endoplasmic reticulum. Biomembranes 2: 95-104. Morre, DJ., Kartenbeck, J. and Franke, W.W. (1979) Membrane flow and interconversions among endomembranes. Biochim. Biophys. Acta 559: 71-152. Munro, S. (1991) Sequences within and adjacent to the transmembrane segment of a2,6-sialyltransferase specify Golgi retention. EMBO J. 10: 3577-3588. Munro, S. (1995) An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J. 14: 4695-4704. Munro, S. and Pelham, H.R (1986) An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46: 291-300. Murata, M., Peranen, 1., Schreiner, R., Wieland, F., Kurzchalia, T.V. and Simons, K. (1995) VIP2l/caveolin is a cholesterol-binding protein. Proc. Natl Acad. Sci. USA 92: 10339-10343. Murphy, C., Saffrich, R., Grummt, W., Gournier, H., Rybin, V., Rubino, M., Auvinen, P., Lutcke, A., Parton, RG. and Zerial, M. (1996) Endosome dynamics regulated by a Rho protein. Nature 384: 427 -432. Narula, N., McMorrow, I., Plopper, G., Doherty, J., Matlin, K.S., Burke, B. and Stow, J.L. (1992) Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes. J. Cell BioI. 117: 27 - 38. Narula, N. and Stow, J.L. (1995) Distinct coated vesicles labelled for p200 bud from trans-Golgi network membranes. Proc. Natl Acad. Sci. USA 92: 2874-2878. Newman, A.P., Graf, J., Mancini, P., Rossi, G., Lian, J.P. and Ferro-Novick, S. (1992) Sec22 and SLY2 are identical. Mol. Cell Bioi. 12: 3663-3664. Niehrs, C. and Huttner, W.E. (1990) Purification and characterization of tyrosylprotein sulfotransferase. EMBO J. 9: 35-42. Nilsson, T., Lucocq, J.M., Mackay, D. and Warren, G. (1991) The membrane spanning domain of ~1,4-galactosyl transferase specifies trans-Golgi localization. EMBO J. 10: 1367-1375. Nilsson, T., Pypaert, M., Hoe, M.H., Slusarevicz, P., Berger, E.G. and Warren, G. (1993a). Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells. J. Cell BioI. 120: 5-13. Nilsson, T., Slusarewicz, P., Hoe, M.H. and Warren, G. (1993b). Kin recognition: a model for the retention of Golgi enzymes. FEBS Lett. 330: 1-4. Nilsson, T., Rabouille, C., Hui, N., Watson, R and Warren, G. (1996) The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells. J. Cell Sci. 109: 1975-1989. Nilsson, T. and Warren, G. (1994) Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus. Curro Opin. Cell BioI. 6: 517-521. Notwehr, S.F., Conibear, E. and Stevens, T.H. (1995) Golgi and vacuolar membrane proteins reach the vacuole in vpsl mutant yeast cells via the plasma membrane. J. Cell BioI. 129: 35-46. Novick, P. and Brennwald, P. (1993) Friends and family: the role of Rab GTPases in vesicular traffic. Cell 75: 597-601. Novick, P., Field, C. and Schekman, R (1980) Identification of 23 complementation groups required for post• translational events in the yeast secretory pathway. Cell 21: 205-215. Novick, P., Garrett, M.D., Brennwald, P., Lauring, A., Finger, F.P., Collins, R and TerBush, D.R. (1995) Control of exocytosis in yeast. Cold Spring Harbor Symp. Quant. BioI. 60: 171-177. Nuoffer, C. and Balch, W.E. (1994) GTPase: Multifunctional molecular switches regulating vesicular traffic. Ann. Rev. Biochem. 63: 949-90. 124 M.G. Farquhar and H.-P. Hauri

Niirnberg, B. and Ahnert-Hilger, G. (1996) Potential roles of heterotrimeric G proteins of the endomembrane system. FEBS Lett. 389: 61-65. Ohno, H., Fournier, M.e., Poy, G. and Bonifacino, J.S. (1996) Structural detenninants of interaction of tyrosine• based sorting signals with the adaptor medium chains. J. BioI. Chern. 271: 29009-29015. Ohno, H., Stewart, J., Fournier, M.C., Bosshart, H., Rhee, I., Miyatake, S., Saito, T., Gallusser, A., Kirchhausen, T. and Bonifaciono, J.S. (1995) Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269: 1872-1875. Orci, L., Palmer, DJ., Amherdt, M. and Rothman, J.E. (1993) Coated vesicles assembly in the Golgi requires only coatomer and ARF proteins from the cytosol. Nature 364: 732-734. Orci, L., Ravazzola, M., Amherdt, M., Perrelet, A., Powell, S.K., Quinn, D.L. and Moore, H.P. (1987) The trans• most cisternae of the Golgi complex: a compartment for sorting of secretory and plasma membrane proteins. Cell 51: 1039-1051. Orci, L., Ravazzola, M., Meda, P., Holcomb, C., Moore, H.-P., Hicke, L. and Schekman, R (1991) Mammalian sec 23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm. Proc. Natl Acad. Sci. USA 88: 8611-8615. Orci, L., Schekman, R. and Perrelet, A. (1996) Interleaflet clear space is reduced in the membrane of COP I and COP II-coated buds/vesicles. Proc. Natl Acad. Sci. USA 93: 8968-8970. Ostennann, J., Orci, L., Tani, K., Amherdt, M., Ravazzola, M., Elazar, Z. and Rothman, J.E. (1993) Stepwise assembly of functionally active transport vesicles. Cell 75: 1015-1025. Padfield, PJ. and Jamieson, J.D. (1991) Low molecular weight GTP-binding proteins associated with zymogen granule membranes from rat pancreas. Biochern. Biophys. Res. Cornrn. 174: 600-605. Palade, G.E. (1953) Fine structure of blood capillaries. J. Appl. Physics 24: 1424, Palade, G.E. (1975) Intracellular aspects of the process ofprotein secretion. Science 189: 347-358. Palade, G.E. (1997) Role of plasmalemmal vesicles in microvascular penneability./n: R G. Crystal and J.B. West (eds): The Lung: Scientific Foundations, Lippincott-Raven Publishers, Philadelphia, pp 673-683. Parton, RG. (1996) Caveolae and caveolins. Curro Opin. Cell BioI. 8: 542-548. Parton, RG., Joggerst, B. and Simons, K. (1994) Regulated internalization of caveolae. J. Cell BioI. 127: 1199• 1215. Pearse, B.M. and Robinson, M.S. (1990) Clathrin, adaptors, and sorting. Ann. Rev. Cell BioI. 6: 151-171. Pelham, H.R.B. (1988) Evidence that luminal ER proteins are sorted from secreted proteins in post-ER compartment. EMBO J. 7: 913-918. Pelham, H.RB. (1989) Control of protein exit from the endoplasmic reticulum. Ann. Rev. Cell BioI. 5: 1-23. Pelham, H.RB. (1991) Recycling of proteins between the endoplasmic reticulum and Golgi complex. Curro Opin. Cell BioI. 3: 585-591. Pelham, H.R.B. (1994) About turn for the COPs? Cell 79: 1125-1127. Pelham, H.RB. (1995) Sorting and retrieval between the endoplasmic reticulum and Golgi apparatus. Curro Opin. Cell BioI. 7: 530-535. Pelham, H.RB., Banfield, D.K. and Lewis, MJ. (1995) SNAREs involved in traffic through the Golgi complex. Cold Spring Harbor Syrnp. Quant. BioI. XL: 105 -II. Pepperkok, R, Scheel, J., Horstmann, H., Hauri, H.-P., Griffiths, G. and Kreis, T.E. (1993) Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell 74: 71• 82. Peter, F., Plutner, H., Zhu, H., Kreis, T.E. and Balch, W.E. (1993) Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro. J. Cell BioI. 122: 1155-1167. Peters, P.J., Hsu, V.W., Ooi, C.E., Finazzi, D., Teal, S.B., Oorschot, V., Donaldson, J. and Klausner, R.D. (1995) Overexpression of wildtype and mutant ARFI and ARF6: Distinct perturbations of nonoverlapping membrane compartinents. J. Cell BioI. 128: 1003-1017. Peyroche, A., Paris, S. and Jackson, C.L. (1996) Nucleotide exchange on ARF mediated by yeast Geal protein. Nature 384: 479-481. Pfeffer, S.R (1994) Rab GTPases: master regulators of membrane trafficking. Curro Opin. Cell BioI. 6: 522- 526. Pfeffer, S.R (1996) Transport vesicle docking: SNAREs and associates. Ann. Rev. Cell Develop. BioI. 12: 441-. 461. Pfeffer, S.R and Rothman, J.E. (1987) Biosynthetic protein transport and sorting by the endoplasmic reticulum and the Golgi apparatus. Ann. Rev. Biochern. 56: 829-852. Pimplikar, S.W. and Simons, K. (1993) Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature 362: 456-458. Pind, S.N., Nuoffer, C., McCaffery, J.M., Plutner, H., Davidson, H.W., Farquhar, M.G. and Balch, W.E. (1994) Rab I and Ca2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to Golgi transport. 1. Cell BioI. 125: 239-252. Pohlmann, R, Boeker, M.W. and von Figura, K. (1995) The two mannose 6-phosphate receptors transport distinct complements of lysosomal proteins. J. BioI. Chern. 270: 27311-27318. Predescu, D., Horvat, R, Predescu, S. and Palade, G.E. (1994) Transcytosis in the continuous endothelium of the myocardial microvasculature is inhibited by N-ethylmaleimide. Proc. Natl Acad. Sci. USA 91: 3014-3018. Protein sorting and vesicular traffic in the Golgi apparatus 125

Predescu, S., Predescu, D. and Palade, G.E. (1997) Plasmalemmal vesicles function as transcytotic carriers for small proteins in the continuous endothelium. Am. J. Physiol. 272: H937-H949. Price, L.S., Norman, J.e., Ridley, A.J. and Koffer, A. (1995) The small GTPases Rac and Rho as regulators of secretion in mast cells. Curro BioI. 5: 68 - 73. Pryer, N.K, Wuestehube, LJ. and Schekman, R (1992) Vesicle-mediated protein sorting. Ann. Rev. Biochem. 61: 471-516. Quinn, P., Griffiths, G. and Warren, G. (1984) Density of newly synthesized plasma membrane proteins in intracel• lular membranes II. Biochemical studies. J. Cell BioI. 98: 2142-2147. Rabouille, C., Hui, N., Kiekbusch, R, Berger, E.G., Graham, G. and Nilsson, T. (1995) Mapping of the distribu• tion of Golgi enzymes involved in the construction of complex oligosaccharides. J. Cell Sci. 108: 1617 -1627. Raposo, G., van Santen, H.M., Leijendekker, R., Geuze, HJ. and Ploegh, H.L. (1995) Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment. J. Cell BioI. 131: 1403-1419. Rizzolo, L.J., Finidori, J., Gonzalez, A., Arpin, M., Ivanov, I.E., Adesnik, M. and Sabatini, D.D. (1985) Biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids. J. Cell BioI. 101: 1351-1362. Robinson, M.S. (1993) The role of clathrin, adaptors and dynarnin in endocytosis. Curro Opin. Cell BioI. 6: 538-544. Robinson, M.S. (1997) Coats and vesicle budding. Trends Cell BioI. 7: 99-102. Robinson, M.S. and Kreis, T.E. (1992) Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effect ofbrefeldin A and G protein activators. Cell 69: 129-138. Roth, J. and Berger, E.G. (1982) Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J. Cell BioI. 93: 223-229. Roth, J., Taatjes, DJ., Lucocq, J.M., Weinstein, J. and Paulson, J.C. (1985) Demonstration of an extensive trans• tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell 43: 287• 295. Roth, J., Taatjes, D.J., Weinstein, J., Pauslon, J.C., Greenwell, P. and Watkins, W.M. (1986) Differential subcom• partmentation of terminal glycosylation in the Golgi apparatus of intestinal absorptive and goblet cells. J. BioI. Chem. 261: 14307-14312. Roth, J., Wang, Y., Eckhardt, A. and Hill, RL. (1994) Subcellular localization of the UDP-N-acetyl-D-galactos• amine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland. Proc. NatlAcad. Sci. USA 91: 8935-8939. Rothberg, K.G., Heuser, J.E., Donzell, W.C., Ying, Y.S., Glenney, J.R. and Anderson, RG. (1992) Caveolin, a protein component of caveolae membrane coats. Cell 68: 673-682. Rothberg, K.G., Ying, Y.S., Kolhouse, J.F., Kamen, B.A. and Anderson, RG. (1990) The glycophospholipid• linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J. Cell BioI. 110: 637 -649. Rothman, J.E. (1994) Mechanisms of intracellular protein transport. Nature 372: 55-61. Rothman, J.E. and Orci, L. (1992) Molecular dissection of the secretory pathway. Nature 355: 409-415. Rothman, J.E. and Orci, L. (1996) Budding vesicles in living cells. Sci. Am. 274: 70-75. Rothman, J.E. and Wieland, F.T. (1996) Protein sorting by transport vesicles. Science 272: 227-234. Rybin, V., Ullrich, 0., Rubino, M., Alexandrov, K., Simon, I., Seabra, C., Goody, Rand Zerial, M. (1996) GTPase activity of Rab5 acts as a timer for endocytic membrane fusion. Nature 383: 266-269. Salama, N.R and Schekman, RW. (1995) The role ofcoat proteins in the bisynthesis of secretory proteins. Curro Opin. Cell Bioi. 7: 536-543. Salamero, J., Sztul, E.S. and Howell, KR (1990) Exocytic transport vesicles generated in vitro from the trans• Golgi network carry both secretory and plasma membrane proteins. Proc. Natl. Acad. Sci USA 87: 7717- 7721. Salpeter, M. and Farquhar, M.G. (1981) High resolution autoradiographic analysis of the secretory pathway in mammotrophs of the rat anterior pituitary. J. Cell BioI. 91: 240-246. Sambrook, J.F. (1990) The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum. Cell 61: 197-199. Sapperstein, S.K., Lupashin, V.V., Schmitt, H.D. and Waters, M.G. (1996) Assembly of the ER to Golgi SNARE complex requires Usolp. J. Cell BioI. 132: 755-767. Saraste, J. and Kuismanen, E. (1992) Pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the Golgi complex. Sem. Cell BioI. 3: 343-355. Saraste, J., Palade, G.E. and Farquhar, M.G. (1987) Antibodies to rat pancreas Golgi subfractions: Identification of a 58 kD cis-Golgi protein. J. Cell Bioi. lOS: 2021-2029. Saraste, J. and Svensson, K (1991) Distribution of the intermediate elements operating in ER to Golgi transport. J. Cell Sci. 100: 415-430. Sargiacomo, M., Sudol, M., Tang, Z. and Lisanti, M.P. (1993) Signal transducing molecules and glycosyl• phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell Bioi. 122: 789-807. 126 M.G. Farquhar and H.-P. Hauri

Satoh, T., Ross, C.A, Villa, A., Supattapone, S., Pozzan, T., Snyder, S.H. and Meldolesi, J. (1990) The inositol 1,4,5-trisphosphate receptor in cerebellar Purkinje cells: Quantitative immunogold labelling reveals concentration in an ER subcompartment. J. Cell BioI. 111: 615-624. Saucan, L. and Palade, G.E. (1994) Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers. J. Cell BioI. 125: 733 - 741. Scheel, AA and Pelham, H.R (1996) Purification and characterization of the human KDEL receptor. Biochemistry 35: 10203-10209. Scheiffele, P., Peranen, J. and Simons, K. (1995) N-glycans as apical sorting signals in epithelial cells. Nature 378: 96-98. Schekman, Rand Orci, L. (1996) Coat proteins and vesicle budding. Science 271: 1526-1533. Schell, M.J., Maurice, M., Stieger, B. and Hubbard, A.L. (1992) 5' nucleotidase is sorted to the apical domain of hepatocytes via an indirect route. J. Cell Bioi. 119: 1173 -1182. Scheller, RH. (1995) Membrane trafficking in the presynaptic nerve terminal. Neuron 14: 893-897. Schiavo, G., Stenbeck, G., Rothman, J.E. and SOIlner, T.H. (1997) Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane tcSNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses. Proc. Natl Acad. Sci. USA 94: 997-1001. Schimmoller, F., Singer-Kriiger, B., SchrOder, S., Kruger, U., Barlow.e, C. and Riezman, H. (1995) The absence of Emp24p, a component of ER-derived COP II-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO J. 14: 1329-1339. Schindler, R, Itin, C., Zerial, M., Lottspeich, E and Hauri, H.-P. (1993) ERGIC-53, a membrane protein of the ER-Golgi intermediate compartment carries an ER retention motif. Eur. J. Cell BioI. 61: 1-9. Schmalzing, G., Richter, H.P., Hansen, A, Schwarz, W., Just, I. and Aktories, K. (1995) Involvement of the GTP binding protein Rho in constitutive endocytosis in Xenopus laevis oocytes. J. Cell BioI. 130: 1319-1332. Schmid, S.L. and Dahrnke, H. (1995) Coated vesicles: a diversity of form and function. FASEB J. 9: 1445 -1453. Schnabel, E., Mains, RE. and Farquhar, M.G. (1989) Proteolytic processing of pro-ACTHlendorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells. Molec. Endocr. 89: 1223-1235. Schnefel, S., Zimmermann, P., Profrock, A, Jahn, R., Aktories, K., Zeuzem, S., Haase, W. and Schulz, I. (1992) High- and small-molecular-weight GTP-binding proteins in zymogen granule membranes of rat pancreatic acinar cells. Cell Physiol. Biochem. 2: 77-89. Schnepf, E. (1993) Golgi apparatus and slime secretion in plants: the early implications and recent models of membrane traffic. Protoplasma 172: 3-11. Schnitzer, J.E., Liu, J. and Oh, P. (1995) Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J. BioI. Chem. 270: 14399-14404. Schnitzer, J.E., Oh, P. and McIntosh, D.P. (1996) Role of GTP hydrolysis in fission of caveolae directly from the plasma membrane. Science 274: 239-242. Schutze, M.-P., Peterson, P.A. and Jackson, M.R (1994) An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum. EMBO J. 13: 1696-1705. Schweizer, A., Clausen, H., van Meer, G. and Hauri, H.-P. (1994) Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylcerarnide synthesis in Vero cells with respect to the endoplasmic reticulum - Golgi intermediate compartment. J. BioI. Chem. 269: 4035-4041. Schweizer, A, Fransen, J.A., Matter, K., Kreis, T.E., Ginsel, L. and Hauri, H.-P. (1990) Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. Eur. 1. Cell BioI. 53: 185-196. Schweizer, A., Fransen, J.AM., Biichi, T., Ginsel, L. and Hauri, H.-P. (1988) Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulovesicular compartment at the cis-side of the Golgi apparatus. J. Cell Bioi. 107: 1643-1653. Schweizer, A., Matter, K., Ketcham, C.M. and Hauri, H.-P. (1991) The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis-Golgi. J. Cell BioI. 113: 45-54. Seaman, M.N., Burd, C.G. and Ernr, S.D. (1996) Receptor signalling and the regulation of endocytic membrane transport. Curro Opin. Cell BioI. 8: 549-556. Seelig, H.P., Schranz, P., Schroter, H., Wiemann, C., Griffiths, G. and Renz, M. (1994) Molecular genetic analyses of a 376-kD Golgi complex membrane protein (Giantin). Molec. Cell BioI. 14: 2564-2576. Serafini, T., Stenbeck, G., Brecht, A, Lottspeich, E, Orci, L., Rothman, J. and Wieland, F.T. (1991) A coat subunit of Golgi-derived nonclathrin coated vesicles with homology to the c1athrin coated vesicle coat protein beta• adaptin. Nature 349: 215-220. Shaper, J.H. and Shaper, N.L. (1992) Enzymes associated with glycosylation. Curro Struct. BioI. 2: 701. Shaywitz, D.A., Orci, L., Ravazzola, M., Swaroop, A. and Kaiser, C. (1995) Human SEC13p functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum. J. Cell BioI. 128: 769-777. Sheff, D., Lowe, M., Kreis, T.E. and Mellman, I. (1996) Biochemical heterogeneity and phosphorylation of coatomer subunits. J. BioI. Chem. 271: 7230- 7236. Simionescu, M., Simionescu, N. and Palade, G.E. (1982) Differentiated microdomains on the luminal surface of capillary endothelium: distribution of lectin receptors. J. Cell BioI. 94: 406-413. Protein sorting and vesicular traffic in the Golgi apparatus 127

Simon, J.-P., Ivanov, I.E., Ren, M., Zeng, J., Shopsin, B., Hersh, D., Tempst, P., Erdjument-Bromage, H., Lui, M., De Lemos-Chiarandini, C., Rosenfeld, M., Gravotta, D., Morimoto, T., Adesnik, M. and Sabatini, D.D. (1995) Regulation of post-Golgi vesicle production in an in vitro system. In: Protein kinesis: The dynamics ofprotein trafficking and stability. Cold Spring Harbor Symposia on Quantitative Biology 60: 179-195. Simon, J.P., Ivanov, I.E., Adesnik, M. and Sabatini, D.D. (l996a). The production ofpost-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity. J. Cell BioI. 135: 355-370. Simon, J.P., Ivanov, I.E., Shopsin, B., Hersh, D., Adesnik, M. and Sabatini, D.D. (1996b). The in vitro generation of post-Golgi vesicles carrying viral envelope glycoproteins requires an ARF-like GTP-binding protein and a protein kinase C associated with the Golgi apparatus. J. BioI. Chern. 271: 16952-16961. Simons, K. and van Meer, G. (1988) Lipid sorting in epithelial cells. Biochemistry 27: 6197-6202. Simons, K. and Virta, H. (1987) Perforated MDCK cells support intracellular transport. EMBO J. 6: 2241-2247. Simons, K. and Zerial, M. (1993) Rab proteins and the road maps for intracellular transport. Neuron II: 789-799. Simpson, F., Bright, N.A., West, M.A., Newman, L.S., Darnell, RB. and Robinson, M.S. (1996) A novel adaptor- related protein complex. J. Cell Bioi. 133: 749-760. Sitia, Rand Meldolesi, J. (1992) Endoplasmic reticulum: a dynamic patchwork of specialized subregions. Malec. BioI. Cell3: 1067 -1072. Sjoberg, E.R and Varki, A. (1993) Kinetic and spatial interrelationships between ganglioside glycosyltransferases and O-acetyltransferase(s) in human melanoma cells. J. BioI. Chern. 168: 10185-10196. Skinner, H.B., McGee, T.P., McMaster, C.R., Fry, M.R., Bell, R.M. and Bankaitis, V.A. (1995) The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline• phosphate cytidylyltransferase activity. Proc. Natl Acad. Sci. USA 92: 112-116. Smart, E.J., Ying, Y.S., Conrad, P.A. and Anderson, R.G. (1994) Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. J. Cell BioI. 127: 1185-1197. Sogaard, M., Tani, K., Ye, RR, Geromanos, S., Tempst, P., Kirchhausen, T., Rothman, J.E. and Sollner, T. (1994) A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell 78: 937-948. Sohn, K., Orci, L., Ravazzola, M., Amherdt, M., Bremser, M., Lottspeich, F., Fiedler, K., Helms, J.B. and Wieland, F.T. (1996) A major transmembrane protein of Golgi-derived COP I-coated vesicles involved in coatomer binding. J. Cell BioI. 135: 1239-1248. Sollner, T., Bennett, M.K., Whiteheart, S.W., Scheller, RH. and Rothman, J.E. (l993a). A protein assembly• disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75: 409-418. Sollner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, S., Geromanos, P., Tempst, P. and Rothman, J.E. (l993b). SNAP receptors implicated in vesicle targeting and fusion. Nature 362: 318-324. Sonnichsen, B., Fiillekrug, J., van Nguyen, P., Diekmann, W., Robinson, D.G. and Mieskes, G. (1994) Retention and retrieval: both mechanisms cooperate to maintain calreticulin in the endoplasmic reticulum. J. Cell Sci. 107: 2705-2717. Stack, J.H., Horazdovsky, B. and Emr, S.D. (1995) Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and GTP-binding proteins. Ann. Rev. Cell Develop. BioI. II: 1-33. Starnnes, M.A., Craighead, M.W., Hoe, M.H., Lampen, N., Geromanos, S., Tempst, P. and Rothman, J.E. (1995) An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding. Proc. Natl Acad. Sci. USA 92: 8011-8015. Stamnes, M.A. and Rothman, J.E. (1993) The binding of AP-I c1athrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein. Cell 73: 999-1005. Stanley, K.K. and Howell, K.E. (1993) TGN38/41: a molecule on the move. Trends Cell BioI. 3: 252-255. Steinhardt, RA., Bi, G. and Alderton, J.M. (1994) Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 263: 390-393. Stenmark, H., Vitale, G., Ullrich, O. and Zerial, M. (1995) Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell 83: 423-432. Stinchcombe, I.e., Nomoto, H., Cutler, D.F. and Hopkins, C.R. (1995) Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex. J. Cell BioI. 131: 1387-1401. Stow, J.L. (1995) Regulation of vesicular transport by GTP-binding proteins. Curro Opin. Nephrol. Hypertension 4: 421-425. Stow, J.L., de Almeida, J.B., Narula, N., Holzman, EJ., Ercolani, L. and Ausiello, D.A. (1991) A heterotrimeric G protein, G-alpha-i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PKI epithelial cells. J. Cell BioI. 114: 1113-1124. Siidhof, T.e. (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375: 645-653. Takei, K., McPherson, P.S., Schmid, S.L. and De Camilli, P. (1995) Dynamin coated tubular membrane invaginations induced by GTP-gS in nerve terminals. Nature 374: 186-190. Tang, B.L., Peter, F., Krijnse-Locker, J., Low, S.H., Griffiths, G. and Hong, W. (1997) The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus. Mol. Cell Bioi. 17: 256-266. Tang, B.L., Wong, S.H., Low, S.H. and Hong, W. (1992) The transmembrane domain ofN-glucosaminyltransferase I contains a Golgi retention signal. J. BioI. Chern. 267: 10122-10126. 128 M.G. Farquhar and H.-P. Hauri

Tang, B.L., Wong, S.H., Qi, XL., Low, S.H. and Hong, W. (1993) Molecular cloning, characterization, subcellular localization and dynamics ofp23, the mammalian KDEL receptor. J. Cell BioI. 120: 325-328. Tatu, U. and Helenius, A. (1997) Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum. J. Cell BioI. 136: 555-565. Teasdale, R.D., D'Agnostaro, G. and Gleeson, P.A. (1992) The signal for Golgi retention of bovine ~1,4 galactosyltransferase is in the transmembrane domain. J. Bioi. Chern. 267: 4084-4096. Teasdale, RD. and Jackson, M.R (1996) Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the Golgi apparatus. Ann. Rev. Cell Develop. BioI. 12: 27-54. TerBusch, D.R and Novick, P. (1995) Sec6, Sec8, and Secl5 are components of a multisubunit complex which localizes to small bud tips in Saccaromyces cervisiae. J. Cell BioI. 130: 299-312. Terui, T., Kahn, RA. and Randazzo, P.A. (1994) Effects of acid phospholipids on nucleotide exchange properties of ADP-ribosylation factor 1. Evidence for specific interaction with phosphatidylinositol 4,5-bisphosphate. J. BioI. Chern. 269: 28130-28235. Ting, A.E. and Scheller, RH. (1995) Insights into the molecular mechanisms of synaptic vesicle docking and fusion. Cold Spring Harbor Syrnp. Quant. Bioi. 60: 361-369. Tooze, S.A. and Huttner, W.B. (1990) Cell-free protein sorting to the regulated and constitutive secretory pathways. Cell 60: 837 -847. Townsley, F. and Pelham, H.RB. (1994) The KKXX signal mediates retrieval of membrane proteins from the Golgi to the ER in yeast. Eur. J. Cell Bioi. 64: 211-216. Traub, L.M. (1997) Clathrin-associated adaptor proteins-putting it all together. Trends Cell BioI. 7: 43-46. Traub, L.M., Kornfeld, S. and Ungewickell, E. (1995) Different domains of the AP-1 adaptor complex are required for Golgi membrane binding and clathrin recruitment. J. BioI. Chern. 270: 4933 -4942. Traub, L.M., Ostrom, J.A. and Kornfeld, S. (1993) Biochemical dissection of AP-1 recruitment onto Golgi membranes. J. Cell Bioi. 123: 561-573. Trowbridge, 1.S., Collawn, J.F. and Hopkins, C.R. (1993) Signal-dependent membrane protein trafficking in the endocytic pathway. Ann. Rev. Cell BioI. 9: 129-161. Ungewickell, E., Ungewickell, H., Holstein, S.E., Linder, R, Prasad, K., Barouch, W., Martin, B., Greene, L.E. and Eisenberg, E. (1995) Role of auxilin in uncoating clathrin-coated vesicles. Nature 378: 632-635. Urrutia, R, Henley, J.R., Cook, T. and McNiven, M.A. (1997) The dynamins: Redundant or distinct functions for an expanding family of related GTPases? Proc. Natl Acad. Sci. USA 94: 377- 384. Vallee, RB. and Okamoto, P.M. (1995) The regulation of endocytosis: identifying dynamin's binding partners. Trends Cell Bioi. 5: 43-47. Van Der Sluijs, P., Hull, M., Zahraoui, A., Tavitian, A., Goud, B. and Mellman, 1. (1991) The small GTP-binding protein rab4 is associated with early endosomes. Proc. Natl Acad. Sci. USA 88: 6313-7. Velasco, A., Hendricks, L., Moreman, K.W., Tulsiani, D.RP., Touster, O. and Farquhar, M.G. (1993) Cell type• dependent variations in the subcellular distribution of a-mannosidase I and II. J. Cell BioI. 122: 39-51. Vertel, B.M., Velasco, A., Lafrance, S., Waters, L. and Kaczman-Daniel, K. (1989) Precursors ofchondroitin sulfate proteog1ycan are segregated within a subcompartment of the chondrocyte endoplasmic reticulum. J. Cell BioI. 109: 1827-1836. Villa, A., Podini, P., Clegg, D.O., Poxxan, T. and Meldo1esi, J. (1991) Intracellular Ca2+ stores in chicken Purkinje neurons: Differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER luminal protein, BiP. J. Cell Bioi. 113: 779-791. Wada, 1., Rindress, D., Cameron, P.H., au, WJ., Doherty, J.J.D., Louvard, D., Bell, A.W., Dignard, D., Thomas, D.Y. and Bergeron, U. (1991) SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. BioI. Chern. 266: 19599-1961O. Wandinger-Ness, A., Bennett, M.K., Antony, C. and Simons, K. (1990) Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J. Cell BioI. Ill: 987-1000. Wang, J., Ladinsky, M.S. and Howell, K.E. (1995) Molecules and vesicle coats involved in the budding of exocytic vesicles from the trans-Golgi network. Cold Spring Harbor Syrnp. Quant. BioI. 60: 139-146. Warnock, D.E. and Schmid, S.L. (1996) Dynamin GTPases, a force-generating molecular switch. BioEssays 18: 885-893. Waters, M.G., Serafini, T. and Rothman, J.E. (1991) Coatomer: a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles. Nature 349: 248-251. Weber, E., Berta, G., Tousson, A., St John, P., Green, M.W., Gopalokrishnan, U., Jilling, T., Sorscher, EJ., Elton, T.S., Abrahamson, DR and Kirk, K. (1994) Expression and polarized targeting of a rab3 isoform in epithelial cells. 1. Cell BioI. 125: 583-94. Weimbs, T., Low, S.H., Chapin, SJ., Whiteheart, S.W., Bennett, M.K. and Mostov, K.E. (1996) The role of the NSF/SNAP/SNARE vesicular fusion machinery in protein targeting in polarized epithelial cells. Malec. BioI. Cell 7: 445a. Weisz, O.A., Swift, A.M. and Machamer, C.E. (1993) Oligomerization of a membrane protein correlates with its retention in the Golgi complex. J. Cell Bioi. 122: 1185-1196. Whitney, I.A. and Kreis, T.E. (1996) Identification of novel coatomer and adaptin-related proteins. Malec. BioI. Cell 7: 79a. Protein sorting and vesicular traffic in the Golgi apparatus 129

Whitney, J.A., Gomez, M., Sheff, D., Kreis, T.E. and Mellman, L (1995) Cytoplasmic coat proteins involved in endosome function. Cell 83: 703-713. Wieland, FT., Gleason, M.L., Serafini, TA. and Rothman, J.E. (1987) The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell 50: 289-300. Wiertz, E.J.H. J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones, TR., Rapoport, TA. and Ploegh, H.L. (1996) Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 384: 432-438. Wilde, A. and Brodsky, F.M. (1996) In vivo phosphorylation of adaptors regulates their interaction with clathrin. J. Cell Bioi. 135: 635-645. Wilson, B.S., Komuro, M. and Farquhar, M.G. (1994) Cellular variations in heterotrimeric G-protein localization and expression in rat pituitary. Endocrinology 134: 233-244. Wong, S.H., Low, S.H. and Hong, W. (1992) The 17-residue transmembrane domain of ~-galactoside a2,6• sialyltransferase is sufficient for Golgi retention. J. Cell BioI. 117: 245-258. Woods, J.W., Goodhouse, J. and Farquhar, M.G. (1989) Transferrin receptors and cation-independent mannose 6• phosphate receptors deliver their ligands to two distinct subpopulations of multivesicular endosomes. Eur. J. Cell BioI. 50: 132-143. Xu, H. and Shields, D. (1993) Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells. J. Cell BioI. 122: 1169• 1184. Yoshimori, T., Keller, P., Roth, M.G. and Simons, K. (1996) Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J. Cell BioI. 133: 247-256. Yuan, L., Barriocanal, J.G., Bonifacino, J.S. and Sandoval, LV. (1987) Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation. J. Cell BioI. 105: 215-227. Zerial, M. and Stenmark, H. (1993) Rab GTPases in vesicular transport. Curro Opin. Cell BioI. 5: 613-620. Zhang, J.Z., Davletov, B.A., Siidhof, T.C. and Anderson, R.G. (1994) Synaptogamin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78: 751-760. Zhao, L., Helms, J.B. and Wieland, FT. (1996) ADP-ribosylation factor interacts directly with coatomer. Malec. Bioi. Cell 7: 599a. Zheng, J., Cahill, S.M., Lemmon, M.A., Fushman, D., Schlessinger, J. and Cowburn, D. (1996) Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity. J. Malec. BioI. 255: 14-21. Zurzolo, c., van't Hof, W., van Meer, G. and Rodriguez-Boulan, E. (1994) VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells. EMBO J. 13: 42-53.