Journal of Cell Science 113, 2331-2343 (2000) 2331 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0780

COMMENTARY Spectrin tethers and mesh in the biosynthetic pathway

Maria Antonietta De Matteis1,* and Jon S. Morrow2,* 1Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy 2Department of Pathology and the Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA *Correspondence may be addressed to either author (e-mail: [email protected]; [email protected])

Published on WWW 14 June 2000

SUMMARY

The paradox of how the Golgi and other organelles can sort membrane proteins. Direct interactions of spectrin with a continuous flux of protein and lipid but maintain actin and centractin (ARP1) provide a link to dynein, temporal and morphological stability remains unresolved. myosin and presumably other motors involved with Recent discoveries highlight a role for the cytoskeleton in intracellular transport. Building on the recognized ability guiding the structure and dynamics of organelles. Perhaps of spectrin to organize macromolecular complexes of one of the more striking, albeit less expected, of these membrane and cytosolic proteins into a multifaceted discoveries is the recognition that a spectrin skeleton scaffold linked to filamentous structural elements (termed associates with many organelles and contributes to the linked mosaics), recent evidence supports a similar role for maintenance of Golgi structure and the efficiency of protein spectrin in organelle function and the secretory pathway. trafficking in the early secretory pathway. Spectrin Two working models accommodate much of the available interacts directly with phosphoinositides and with data: the Golgi mesh hypothesis and the spectrin ankyrin membrane proteins. The small GTPase ARF, a key player adapter protein tethering system (SAATS) hypothesis. in Golgi dynamics, regulates the assembly of the Golgi spectrin skeleton through its ability to control Key words: Cytoskeleton, Cargo selection, Linked mosaic, Micro- phosphoinositide levels in Golgi membranes, whereas domain, Actin, Protein sorting, Ankyrin, ARF, SAATS, Golgi adapter molecules such as ankyrin link spectrin to other scaffold

INTRODUCTION are themselves organized, stabilized, shaped, or linked to the motors of transport. A fundamental and striking property of eukaryotic cells is their Spectrin is a cytoskeletal protein that can control membrane ability to dynamically organize and maintain morphologically organization, stability and shape, and link membranes to the distinct membrane-bounded compartments while selectively motors of transport as well as to all major filament systems. transporting newly synthesized or recycled lipids and proteins Spectrin has also recently emerged as a participant in the through secretory and endocytic pathways. Recent discoveries secretory pathway (for earlier reviews, see Beck and Nelson, attribute an increasingly important role to cytoskeletal proteins 1998; De Matteis and Morrow, 1998). Various isoforms of in this process. Cytoskeletal proteins serve two fundamental spectrin and its key adapter protein ankyrin are present on the functions in membrane trafficking: organelle motility (in Golgi, on transport intermediates, and on the membranes of the conjunction with motor proteins), and membrane-organization endo-lysosomal pathway. Disruption of spectrin function either (in conjunction with adapter and integral membrane proteins). by dominant negative spectrin mutants or by anti-spectrin The outlines of how some cytoskeletal elements contribute to antibodies blocks anterograde transport in the secretory pathway, the motility of organelles or transport intermediates along the and direct regulation of the interaction between spectrin and endocytic and exocytic pathways are well described – for Golgi membranes by the small G protein ADP-ribosylation example, the way that the microtubule system and its factor (ARF) is well established. Although much of the work on associated motors mediate long-distance movement (Holleran spectrin in the secretory and endocytic pathways is recent, and and Holzbaur, 1998; Lippincott-Schwartz, 1998), or the way some has only appeared in abstract form, consideration of these that the actin system and actin-based motors effect shorter- findings in the light of spectrin’s well-studied role at the plasma range movement (Stow et al., 1998, Allan and Schroer, 1999; membrane yields a comprehensible picture of the unique Fath et al., 1994; Heimann et al., 1999). However, much less properties that spectrin brings to the Golgi and other organelles. is known about the way that organelles or transport containers These considerations are the focus of this review. 2332 M. A. De Matteis and J. S. Morrow

THE SPECTRIN MEMBRANE SKELETON: loop, variations in the length of the B helix and variations in EXTENSIBLE LINKED MOSAICS the degree of super coiling appear to control both the flexibility of the link and the overall length of the molecule (Grum et al., First identified as the supporting infrastructure of the plasma 1999). Thus, a more complete concept of the spectrin skeleton membrane of erythrocytes, spectrin is now recognized as the is as a series of extensible linked mosaics (Fig. 1C). This most central player in a ubiquitous and complex linkage structure can accommodate limited membrane deformation, between membranes and the cytosol. By binding limited diffusional motion and the variable spacing required to simultaneously to integral membrane proteins, cytosolic capture a variety of ligands, while adjusting to variations in proteins and certain phospholipids, either directly or through membrane curvature and providing larger-scale order and adapter proteins, spectrin creates a multifunctional scaffold at support. the membrane interface on which macromolecular complexes Finally, at least at the plasma membrane, all interactions of membrane proteins, cytoplasmic signaling molecules, and involving spectrin and its adapter molecules, including the structural elements are organized (Fig. 1). In addition, spectrin elasticity of spectrin itself, appear to be regulated post- binds (either directly or via adapter or motor proteins) to all translationally. Identified mechanisms include phosphorylation major filament systems and thereby links mosaics or islands by a variety of kinases, the action of calcium and calmodulin, of membrane and cytosolic proteins to cytoskeletal elements. calcium-activated proteolysis, and regulation by small GTP- These properties have led to the general concept of the spectrin binding proteins, pH and myristolyation. In addition, multiple membrane skeleton as a series of ‘linked mosaics’ (De Matteis isoforms of spectrin exist (Tables 1 and 2), as do variants that and Morrow, 1998; Morrow et al., 1997). The characteristics arise by alternative mRNA splicing that specifically alter the of the skeleton may vary: in the archetypal erythrocyte helix B-C loop and possibly thereby the flexibility or spacing skeleton, the mosaics are frequent, homogeneous, and joined of a sub-region of the molecule (Cianci et al., 1999b). by short actin filaments so as to form a homogeneous quasi- hexagonal lattice. Alternatively, the mosaics can be sparse and linked to longer microfilaments or to microtubules via HOW DOES THE SPECTRIN SKELETON CONTROL dynein/dynactin, forming the types of spectrin skeleton MEMBRANE STABILITY AND SHAPE? associated with organized receptor clusters or membrane microdomains. Examples of such structures include the Before considering the role of spectrin at the Golgi and in the spectrin skeleton found at the neuronal post-synaptic density secretory pathway, it is worthwhile to ponder its role in the red (Malchiodi-Albedi et al., 1993; Wechsler and Teichberg, cell. Specifically, why is spectrin needed to stabilize the 1998), at the nodes of Ranvier (S. Berghs et al., unpublished; membrane, and why in its absence do red cell membranes Lambert et al., 1997), at the acetylcholine receptor cluster of spontaneously endovesiculate? Early concepts attributed a skeletal muscle (Pumplin, 1995), at the basolateral infoldings of epithelial cells (Drenckhahn and Merte, 1987), and perhaps Fig. 1. Spectrin is a multifunctional extended molecule that forms a the spectrin complex found on tubular-vesicular transport membrane skeleton composed of extensible linked mosaics. (A) Spectrin usually exists as anti-parallel heterodimers of an α- intermediates, the Golgi and other organelles (see below). β β But there is more to the spectrin skeleton. A separate concept spectrin and a -spectrin. Homopolymeric -spectrin complexes exist in skeletal muscle (Bloch and Morrow, 1989) and possibly the that evolved from studies of the erythrocyte identifies spectrin Golgi (Beck et al., 1994; Devarajan et al., 1996). Each subunit as a type of molecular spring or shock-absorber, a molecule displays a tripartite organization; non-homologous ends (regions 1 that not only tethers integral and cytosolic proteins but also and 3) flank a central domain (region 2) composed of multiple ≈106- controls their spacing by altering its flexibility and contour residue coiled-coil α-helical repeats. Functional specializations length. Previously, this notion found support only in appear within these repeats as indicated. Ligands reported to bind biophysical studies that detected altered elasticity of the one or more forms of spectrin, and the best-recognized adapter membrane as a function of spectrin perturbation (Stokke et al., proteins, are shown along with their approximate binding site. 1986), in the significant differences in scaled flexural rigidity (B) The ‘extensible linked mosaic’ model of spectrin action. The (a measure of stiffness) between different types of spectrin fundamental role of the spectrin skeleton is to control lateral order in (Coleman et al., 1989), and in morphological studies that the plane of the membrane (see Fig. 2). Through its capacity to bind multiple ligands selectively and to self-associate through hetero- and revealed the in situ length of spectrin to be variable and homo-typic interactions, end on and side to side, spectrin can form much shorter than the 2000-Å length of extended spectrin ordered arrays or mosaics of limited size. Associated with these heterodimers (McGough and Josephs, 1990; Ursitti et al., nascent arrays are embedded and soluble proteins. The ability of 1991). Grum et al. (1999) have now elegantly revealed the spectrin to expand or contract, and to alter its flexibility, controls the structural basis of this unusual property. A defining feature of density of packing within each mosaic and possibly local membrane spectrin and spectrin-related proteins (including α-actinin and curvature. Mosaics are joined by linking interactions involving F- dystrophin) is the presence of many tandem, antiparallel actin, microtubules or intermediate filaments to form larger arrays. coiled-coil repeats. Whereas the features of a single repeat are Active redistribution or macro-organization of such mosaics along predictable on the basis of its α-helical content and have been microfilaments or microtubules is achieved by linkage of the mosaics confirmed (Yan et al., 1993), it is the linker region between to the appropriate motors. (C) The basic structural motif of spectrin is a series of tandem, antiparallel coiled-coil repeats. Two repeats are repeats and the ability of two spectrin chains to supercoil that joined by a continuation of helix C into helix A of the adjacent appears to control the length and flexibility of spectrin (Fig. repeat. Conformational rearrangements involving the helix B-C loop, 1B). and variations in super-coiling between the two spectrin chains, can Each spectrin repeat is composed of three helices (A, B and rapidly vary the length and flexibility of the molecule (adapted with C). Conformational rearrangements involving the helix B-C permission from Grum et al., 1999). Spectrin in the secretory pathway 2333 2334 M. A. De Matteis and J. S. Morrow

Table 1. Summary of spectrin isoforms Spectrin Human chromosome Pre-Golgi Golgi TGN/endosome/lysosome Plasma membrane Comments αΙ 1 No No Yes Yes Predominant in red cells; also endo/lysosomal compartment αΙΙ 9 No No No Yes Generalized plasma membrane α-spectrin βΙ 14 Yes Yes Yes Yes Predominant in red cells (βΙΣ1), also in brain and muscle (βΙΣ2); immunoreactive with Golgi forms βΙΙ 2 No No No Yes Generalized plasma membrane β-spectrin βΙΙΙ 11 Yes Yes Yes No (trace) Golgi and transport container associated spectrin; binds ARP1 and munc13 βΙV 19 No peri-nuclear Yes Yes MAD2 domain contains novel ICA/512 secretory granule protein binding domain; associates also with nodes of Ranvier βV 15 ND ND ND Yes Preserved actin binding and MAD1 domains (Stabach and Morrow, 2000); associates with internal membranes; orthologue of Drosophila beta-heavy

All spectrins have a tripartite structure composed of non-homologous regions 1 and 3 at their termini and a central region 2 composed of a variable number of ≈106-residue repeat units (reviewed by Morrow, 1998). Two genes encode α-spectrins that characteristically display an N-terminal β-spectrin-binding site, a central SH3 domain, and two C-terminal calcium-binding EF-hand motifs. Five genes exist that encode β-spectrins.

Table 2. Summary of ankyrin isoforms Ankyrin Human chromosome Pre-Golgi Golgi TGN/endosome/lysosome Plasma membrane Comments Ank R 8 210 No No No Yes Predominant red cell form. 24 repeat units. May be a basolateral plasma membrane- associated ankyrin in MDCK cells SR Yes ?? No No Unusually small transcript of 3′ exon found in skeletal muscle sarcoplasmic reticulum 195 ?? ?? Yes Yes No Not characterized; immunologically similar to AnkR AnkB 4 Required for intracellular vesicular sorting 440 No No Yes Yes 24 repeats; large non-homologous insertion 220 No No Yes Yes 24 repeats AnkG 10 Many isoforms arise by alternative mRNA splicing. Most widely expressed ankyrin family 480 No No No Yes 24 repeats; large non-homologous insertion 270 No No Yes Yes 24 repeats; non-homologous insertion 190 No No Yes Yes 24 repeats 119 Yes Yes Yes No 13 repeats; truncated regulatory domain 120 No No Yes No 4 repeats 100 No No Yes No 0 repeats; preserved spectrin- binding/regulatory domains only

Three ankyrin genes are known, and the encoded proteins display three domains: (i) a highly-conserved N-terminal domain of tandemly arrayed repeats (33- residues each) that bind many proteins; (ii) a well-conserved central domain that binds spectrin; and (iii) a variably sized C-terminal ‘regulatory’ domain that can modulate the activities of the first two domains. Several novel ankyrin isoforms, some of which lack all or part of the repetitive or regulatory domain, or have large inserts, have been described. mechanical role to spectrin, arguing that it provided the the primary role of spectrin is not to lend rigidity to the increment of elasticity and rigidity needed to withstand the membrane directly, but rather to control the lateral distribution turbulence of circulation. A better model focuses on how of integral membrane proteins. In this view, alterations in integral proteins themselves affect membrane stability and membrane shape and stability arise from processes that affect shape. For example, in hereditary ovalocytosis, a nine-residue the distribution of integral membrane proteins. Examples of deletion in the red cell anion-exchange protein AE1 does not such processes include the weakening of contacts between the alter the structure or composition of the spectrin skeleton, but skeleton and membrane proteins (as in many forms of yields rigid, ovalocytic red cells (Liu et al., 1995; Mohandas hereditary spherocytosis), the weakening of lateral associations et al., 1992). Similarly, deletion of the AE1 gene yields fragile within the skeleton (as in hereditary elliptocytosis), or red cells but does not change the spectrin skeleton or its alterations in the abundance or properties of the integral association with the bilayer (Peters et al., 1996; Southgate et membrane proteins themselves (as in ovalocytosis). al., 1996). Experimentally, the elasticity of the membrane A more rigorous way to view the role of a peripheral depends as much on integral membrane protein attachments as membrane skeleton and its relationship to integral membrane on any property of the skeletal lattice itself (Sleep et al., 1999). protein distributions is to consider a biological membrane as a These seemingly disparate observations can be reconciled if trilayer consisting of a skeletal layer (the spectrin skeleton, or Spectrin in the secretory pathway 2335 any coat complex, such as coat protein (COP) I or II or clathrin) THE COMPOSITION AND ASSEMBLY OF THE in close contact with a phospholipid bilayer with its embedded GOLGI-ASSOCIATED SPECTRIN SKELETON proteins (Kralj-Iglic et al., 1996; Mohandas and Evans, 1994; Svetina et al., 1996; reviewed by Morrow et al., 1997). The two Spectrin lipid layers are joined by hydrophobic forces; the skeleton is Although many studies have detected spectrin or other attached, directly or via adapter proteins, through interactions components of the spectrin skeleton in association with with integral membrane proteins or lipids. The energy organelles (e.g. see Black et al., 1988; De Cesaris et al., 1989; contributed by the embedded proteins is proportional to their Malchiodi-Albedi et al., 1993; Riederer et al., 1986; Zagon et abundance, the curvature of the membrane, and the strength of al., 1986), the exact nature of these associations has only their interactions with the surrounding bilayer and other recently been revealed (Beck et al., 1994, 1997; Beck and proteins in the bilayer (Kralj-Iglic et al., 1996). At equilibrium, Nelson, 1996, 1998; De Matteis and Morrow, 1998; Devarajan the energy of this system will seek a minimum, and many and Morrow, 1996; Devarajan et al., 1996; Godi et al., 1998; integral membrane proteins will seek to aggregate or be Hoock et al., 1997; Stankewich et al., 1998; Ziemnicka-Kotula excluded from regions of increased membrane curvature. The et al., 1998). Most antibodies to erythrocyte βΙ spectrin result of this tendency is that membrane fragmentation and variably mark by indirect immunofluorescence a perinuclear vesiculation become energetically preferred as the reticular complex coincident with resident Golgi markers concentration of protein in the membrane increases (Fig. 2). (Beck et al., 1994; Devarajan and Morrow, 1996; Godi et al., This tendency is resisted by the favorable energetics of 1998). Such antibodies also often stain punctate 500-nm-and- attaching integral proteins to a homogeneous skeleton. smaller cytoplasmic structures that partially coincide with Conversely, discontinuities in the skeleton may favor local markers of both pre-Golgi as well as post-Golgi compartments inhomogeneities, creating specialized microdomains that can (see below; P. Devarajan and J. S. Morrow, and Y. Ch’ng, promote curvature, shape variation, and even vesiculation. unpublished; Devarajan and Morrow, 1996; Stankewich et al., Thus, although not all integral membrane proteins will require 1998). By immunoprecipitation or western blotting, antibodies active management by a peripheral skeleton (as born out by the to βΙ spectrin detect a 220–240-kDa protein in most cultured rapid diffusional mobility of some resident Golgi proteins, see cell lines. Immunoelectron microscopy confirms the presence Cole et al., 1996), one can anticipate that many will – perhaps of spectrin on the Golgi (Beck et al., 1994), although those that are more complex, multimeric or bulky. refinement of its ultrastructural characterization has proven A necessary feature of above model is that spectrin must surprisingly difficult, and the question of whether specific bind, either directly or indirectly, to many integral membrane types of spectrin are confined to any specific subcompartment proteins (see Fig. 1). How can so many different ligands be of the Golgi remains open. Disruption of the Golgi by brefeldin bound with specificity? Beyond the fact that spectrin is a large A (BFA) disrupts the Golgi-associated spectrin (Beck et al., and extended molecule, one answer appears to lie in the 1994; Godi et al., 1998), whereas spectrin remains with the remarkable properties of ankyrin, spectrin’s best-understood residual Golgi elements following nocodazole dispersal (Beck adapter. With few exceptions (Table 2), ankyrins typically et al., 1994). Taken together, these results indicate that a βΙ- possess a well-conserved spectrin-binding domain coupled to a spectrin-related protein is dynamically associated with the variable number of 33-residue homologous repeat units. The Golgi complex in many (if not all) cells. ankyrin repeat is a generalized protein-protein interaction motif Sequences derived from βΙ spectrin can also associate with found in many proteins (for review, see Michaely and Bennett, the Golgi and function in (or block) the secretory pathway. 1992). Each ankyrin repeat forms an L-shaped structure Full-length βΙΣ1 spectrin (Beck et al., 1994) and recombinant consisting of a β-hairpin and two α-helices; multiple repeats peptides representing specific βΙΣ1 and βΙΣ2 spectrin assemble to form an oblong structure in which their α-helical sequences bind to the Golgi in cultured MDCK cells cores are shielded and their β-hairpins are exposed (e.g. see (Devarajan et al., 1997a). These studies identify a specific Foord et al., 1999; Gorina and Pavletich, 1996; Venkataramani constitutive Golgi-targeting signal near the N-terminus of βΙ et al., 1998; Yang et al., 1998; Fig. 3). Sequence alignment of spectrin; this sequence includes the actin-binding domain and the repeat units also reveals that the exposed loops vary membrane-association domain 1 (MAD1; Davis and Bennett, markedly, whereas the helices are conserved. The analogy that 1994; Lombardo et al., 1994). Similar constructs derived from comes to mind is that of a Velcro® ball, a multitude of potential βΙΙ spectrin sort not to the Golgi but to the plasma membrane protein-binding sites being created by the bristly surface of the (Stabach et al., 1993; Stabach et al., unpublished). A second highly variable hairpin loops. For example, AnkG119, the form powerful signal that directs the association of spectrin with the of ankyrin associated with the Golgi and pre-Golgi Golgi is membrane-association domain 2 (MAD2), which intermediates, has 13 ankyrin-repeat units (Devarajan et al., includes a pleckstrin homology (PH) domain. Although some 1996). If one assumes that each of the β-hairpin loops can spectrins lack this domain (e.g. βΙΣ1 from erythrocytes), most provide only a single interaction site (a very conservative possess it (summarized in Table 1). assumption), and that an effective interaction with a protein The MAD2 sequence varies greatly between different ligand is generated by any unique combination of three hairpin spectrins in this domain (Berghs et al., 1999; Stankewich et al., loops, then AnkG119 could offer unique binding sites to >1700 1998; Stabach and Morrow, 2000), which presumably reflects different proteins (one at a time, of course). Although the true functional specialization. At least six MAD2 ligands have been valency of ankyrin is unlikely to be so high, the potential of this identified for different spectrins: βγ G protein signaling single family of adapter molecules to bind so many diverse subunits (Wang et al., 1994); D3BP, a very large protein ligands is an important consideration as one seeks to understand (>400 kDa) of unknown function (Cianci et al., 1997; Li et al., the role of these proteins in membrane biogenesis and secretion. 1998); phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2; 2336 M. A. De Matteis and J. S. Morrow

Fig. 2. Organization of integral membrane proteins by a peripheral skeleton stabilizes biological membranes. Integral proteins can spontaneously distort a bilayer membrane as they seek states of lower energy, causing membrane instability and vesiculation. This process is ameliorated or controlled if their distribution is managed by a peripheral skeleton.

Hyvonen et al., 1995); the GLUT4 receptor (Corcoran et al., 1997); fodaxin (A60), an axon-specific protein (Hayes et al., 1997); and possibly (although not yet proven) islet cell autoantigen (ICA) 512/IA-2, a receptor tyrosine phosphatase associated with secretory granules in neuroendocrine cells (Berghs et al., 1999, and unpublished). The first three of these ligands bind to the spectrin PH domain; the others do not. Additional ligands no doubt associate with this domain, and this is also the site targeted by several kinases (Fig. 1). Although transfection of peptides that have the MAD2 of βΙΣ2 spectrin reveals that this domain alone does not concentrate in the Golgi (Devarajan et al., 1997a), in vitro studies reveal that it modulates the association of spectrin with isolated Golgi fractions (Godi et al., 1998) and that binding is regulated by the small GTPase ARF, a crucial player in Golgi dynamics. The role of ARF in the regulation of spectrin dynamics in the secretory pathway is particularly interesting. As noted above, BFA, a fungal toxin that prevents the activation of ARF, induces the rapid release of spectrin from Golgi membranes (Godi et al., 1998), an action directly related to the ability of ARF to enhance the synthesis of PtdIns(4,5)P2 on these membranes (Godi et al., 1998, 1999). The effect of ARF on PtdIns(4,5)P2 synthesis is independent of its ability to stimulate phospholipase D (PLD) or the assembly of COPI. In fact ARF is able to recruit and maintain a specific PI4K isoform, PI4Kβ, and an unidentified PtdInsP5 kinase on Golgi membranes (Godi et al., 1999). The loss of PI4Kβ activity, obtained by transfecting the dominant negative mutant D656A-PI4Kβ (which is devoid of kinase activity), induces a tubulovesicular transformation of the Golgi complex. Phosphoinositides such as PtdIns(4,5)P2 thus appear to play both a direct structural role in Golgi membranes, and act as binding sites for

Fig. 3. The repeat structure of ankyrin forms a polyvalent generalized protein-protein interaction motif. The p53-binding protein (p53bp) contains four ankyrin-repeat units; its three-dimensional structure reveals the basic features of this motif (top left; Gorina and Pavletich, 1996). Each repeat contains two short helices and an exposed β-hairpin (arrow). Ligands interact at two or three points on or between the protruding hairpin loops. In vitro binding studies have identified short (7-25 residue) sequences within at least five proteins that appear to mediate binding (summarized in Zhang et al., 1998). The structure of one of these short ankyrin-binding sequences, the ankyrin-binding domain in α-Na+/K+ ATPase, is shown. This motif forms an exposed loop on a β-stalk, which is hypothesized to interact with one or two ankyrin repeats as depicted in the cartoon (adapted with permission from Zhang et al., 1998). Spectrin in the secretory pathway 2337 important regulatory and structural proteins, including Finally, a Golgi-associated α-spectrin has not been detected. spectrin. The ARF-induced generation of PtdIns(4,5)P2 Whether this is due to poor cross-reactivity of existing appears to be a key mechanism by which ARF regulates the antibodies to α-spectrin with a Golgi α-spectrin, or whether all recruitment of spectrin to Golgi membranes, whereas reduced Golgi-associated spectrins are exclusively β type, remains to PtdIns(4,5)P2 availability (for example, as induced by be determined. A precedent for the formation of antiparallel PtdIns(4,5)P2-chelating agents or inhibitors of PtdIns(4,5)P2 homopolymeric β-spectrin complexes exists: such structures synthesis), strongly inhibits the ability of ARF to promote reside beneath acetylcholine receptor clusters in skeletal spectrin assembly on the Golgi (Godi et al., 1998). muscle (Bloch and Morrow, 1989; Pumplin, 1995). Yet, the Beyond stimulating the association of spectrin with isolated sequence of βΙΙΙ spectrin suggests that it can bind to α-spectrin Golgi fractions, ARF dependent increases in PtdIns(4,5)P2 (Stankewich et al., 1998), and two-hybrid analysis (M. C. levels also stimulate the recruitment to Golgi membranes of a Stankewich and J. S. Morrow, unpublished) and the co- discrete set of other cytosolic proteins, including ankyrin, actin, immunoprecipitation of βΙΙΙ spectrin with α-spectrin confirm a 230-kDa protein, a 170-kDa protein, a 100-kDa protein, and that the two do associate, at least under some conditions a 30-kDa protein. This set of proteins does not include (Cianci et al., 1999a). However, there are currently no specific coatomer components and is presumably recruited as a candidates for a Golgi-associated α-spectrin. complex with spectrin, given that it dissociates en-bloc from the Golgi in the presence of anti-spectrin agents (Godi et al., Ankyrin 1998). Although other interactions between spectrin and the A second major component of the Golgi spectrin skeleton is Golgi also exist, including those mediated by adapter proteins the adapter protein ankyrin. Three ankyrin genes are and the broad MAD3 region of spectrin (Y. Ch’ng et al., recognized (Table 2), and multiple forms arise by alternative unpublished; Devarajan et al., 1997a), the model that emerges mRNA splicing. The Golgi-associated ankyrin isoform, is that the synergistic action of at least two binding domains – AnkG119, is the only Golgi-associated ankyrin that has been one constitutive (MAD1), the other ARF regulated (MAD2) – completely characterized. Immunofluorescence and cell drives the assembly and stabilization of spectrin onto the Golgi. fractionation reveal that this ANK3 family product is tightly co- However, and despite the immunological and functional localized with the Golgi and possibly the ERGIC in MDCK similarity between the Golgi-associated spectrin and βΙΣ2 and COS cells (Devarajan et al., 1996; Stankewich et al., 1998), spectrin, βΙ spectrin transcripts per se have not been detected and co-precipitates with Golgi-associated spectrin (Devarajan in cultured MDCK cells even by sensitive RT-PCR et al., 1997a, 1998; Godi et al., 1998). Transcripts of other methodologies (P. R. Stabach, Y. Ch’ng and J. S. Morrow, ANK3 family members, including 120-kDa and 100-kDa unpublished). In addition, not all antibodies to βΙ spectrin isoforms, have been identified with late endolysosomes in recognize the Golgi-associated protein (P. Devarajan and J. S. macrophages (Hoock et al., 1997). These isoforms differ from Morrow, P. Marra and M. A. De Matteis, unpublished AnkG119 in that they lack most of the N-terminal repeat region observations). We therefore originally designated the Golgi but do have spectrin-binding and regulatory domains spectrin as βΙΣ∗. It has now been identified as βΙΙΙ spectrin characteristic of the full-sized ankyrins. Finally, a 195-kDa (Stankewich et al., 1998). This novel spectrin, which ankyrin that co-localizes with the trans-Golgi (TG) and trans- Sakaguchi et al. (1998) independently identified as a ligand Golgi network (TGN) has been detected on the basis of for munc13 (a component of the neurotransmitter-release immunologic criteria (Beck et al., 1997). Although the size of machinery of the presynaptic terminal), cross-reacts with many this ankyrin is very similar to that of the kidney plasma antibodies to βΙ spectrin, co-localizes and co-fractionates with membrane AnkG190 (Thevananther et al., 1998), its reactivity Golgi markers, and exerts even more profound effects on towards antibodies to AnkR and its localization in the Golgi secretory pathway function when transfected into cultured cells suggest that it is yet another, and possibly unique, form of than do βΙ spectrin peptides (Y. Ch’ng et al., unpublished). ankyrin. Collectively, the finding of distinct ankyrins in However, only a fraction of the total cellular βΙΙΙ spectrin is secretory, endocytic/lysosomal and plasma membrane associated with the Golgi; the rest is associated with other compartments suggests that this protein family plays a role membrane fractions (Stankewich et al., 1998). Whether the in maintaining the distinct membrane profiles of these subtle differences between the staining patterns obtained with compartments. antibodies to βΙ and antibodies to βΙΙΙ spectrin are a consequence only of differing antibody sensitivities and Actin epitope preferences, or whether other undiscovered spectrins A major filament system linking spectrin complexes at the also associate with the Golgi, remains an open question. In this plasma membrane is actin. Although many actin-binding regard, it is noteworthy that two additional spectrins were proteins are associated with the Golgi (Weiner et al., 1993; recently identified, βIV spectrin (Berghs et al., 1999) and βV McCallum et al., 1998; Heimann et al., 1999), the presence of spectrin (Stabach and Morrow, 2000; Table 1). These initial actin itself on the Golgi has been controversial. Recent in vivo reports identify βIV spectrin in association with the nodes of and in vitro studies make the case for Golgi-associated actin Ranvier and possibly with secretory granules, and βV spectrin and indicate that spectrin provides at least one means by which as the human orthologue of Drosophila β-heavy spectrin. In actin binds this compartment. Golgi morphology is sensitive to mammalian cells, βV spectrin associates with specialized perturbations that disrupt or modify actin (Babia et al., 1999; internal membrane structures such as the photoreceptor disks di Campli et al., 1999). Actin can be co-isolated with Golgi of the retinal outer segment (Stabach and Morrow, 2000). membranes from tissues and cells, and it associates with Whether these new spectrins play any direct role in the Golgi isolated salt-washed Golgi membranes in vitro (Fath and or the secretory or endocytic pathways is unknown. Burgess, 1993; Godi et al., 1998). Light and electron 2338 M. A. De Matteis and J. S. Morrow microscopy show that actin filaments are present on the Golgi dominant negative inhibitors of the transport of certain (Heimann et al., 1999; Valderrama et al., 2000). The membrane proteins to the plasma membrane. In particular, association of actin with Golgi membranes in vitro is ATP when peptides containing this region were expressed in MDCK dependent, stimulated by ARF, and partially inhibited by the cells, Na+/K+-ATPase accumulates in the endoplasmic same spectrin-derived polypeptides that interfere with the reticulum, and the glycosylation of its β-chain, which occurs assembly of spectrin onto the Golgi (Godi et al., 1998). The in the Golgi, is impaired (Devarajan et al., 1997a). The same molar ratio between actin and spectrin (10:1) on Golgi recombinant βΙ spectrin peptides, as well as anti-spectrin membranes is higher than that measured at the plasma antibodies, inhibit the transport of vesicular stomatitis virus membrane (7:1), which suggests that the actin filaments of the (VSV) G protein from the endoplasmic reticulum to the Golgi spectrin-based skeleton at the Golgi are either longer than those when they are introduced into semi-intact VSV-infected NRK at the plasma membrane or that spectrin-independent binding cells. The inhibition of transport of vesicular stomatitis virus sites provided by other actin-binding proteins also contribute G protein (VSV-G) to the Golgi persists even when the peptides (Heimann et al., 1999). are added after a 15°C temperature block, a manipulation that causes protein exiting from the ER to accumulate in the Centractin (ARP1) intermediate compartment (Godi et al., 1998). These data The actin-related protein ARP1 (centractin) binds to Golgi- indicate that the spectrin skeleton must exert its role in the early associated spectrin in vitro, in yeast two-hybrid screens and in secretory pathway, at the ER-to-Golgi interface. Time-lapse vivo (Devarajan et al., 1997b; Holleran et al., 1996; E. A. video microscopy of COS cells transfected with green Holleran et al., unpublished). ARP1 is a component of fluorescent protein (GFP)-tagged βΙΙΙ spectrin peptides dynactin, a multimolecular complex required for the dynein demonstrates that spectrin indeed decorates tubular-vesicular activity that contributes to intracellular vesicular motility and transport intermediates moving along microtubules between the positioning of the Golgi (see below). the ER and the Golgi, and that spectrin’s region-I/MAD1 sequences are required for this movement (Y. Ch’ng et al., Adducin unpublished). These studies also show that ERGIC53 (a marker Additional proteins first identified as part of the erythrocyte of the intermediate compartment) and GFP-tagged VSV-G spectrin skeleton might also contribute to the Golgi skeleton, protein co-localize with a fraction of βΙΙΙ spectrin in the pre- although at present the available data is incomplete. Adducin Golgi compartment in COS cells (Y. Ch’ng et al., unpublished). is a heterodimeric, calmodulin-, PKC-, and Rho-kinase- Several studies have also documented that ankyrin has a regulated actin and spectrin cross-linking protein that functional role in the ER exit and proper sorting of many precipitates with ARP1 and spectrin complexes in cultured membrane proteins, including the anion exchanger AE1 cells (Holleran et al., 1996). Immunofluorescence studies (Gomez and Morgans, 1993), Na+/K+-ATPase (Devarajan et indicate that some anti-adducin antibodies co-localize with al., 1997a), Ca-ATPase and the ryanodine receptor (Tuvia et Golgi-specific markers in MDCK and COS cells (M. C. al., 1999), and the lymphocyte tyrosine phosphate phosphatase Stankewich and J. S. Morrow, unpublished observations) and CD45 (Pradhan and Morrow, 1999). In at least two of these in NRK cells (M. A. De Matteis and G. Bianchi, unpublished). cases (Na+/K+-ATPase and CD45) this requirement for ankyrin early in the secretory pathway relates to its ability to link these integral proteins to spectrin (P. Devarajan et al., unpublished; THE ROLE OF SPECTRIN IN MEMBRANE Pradhan and Morrow, 1999). Interestingly, although the exit of TRAFFICKING another protein from the ER, E-cadherin, is not ankyrin dependent (Devarajan et al., 1997a, 2000), entry of the protein As noted above, spectrin has been identified on many into the secretory pathway does require β-catenin, a different intracellular membranes besides the Golgi, including type of putative adapter protein (Chen et al., 1999), which, at chromaffin granules, synaptic vesicles, the endoplasmic the plasma membrane, links spectrin and actin to E-cadherin reticulum, the nuclear membrane, perinuclear Golgi-like via α-catenin (Nelson and Hammerton, 1989; Provost and vesicles, organelles, and as part of a cytoplasmic meshwork Rimm, 1999; D. Pradhan et al., unpublished; Roe et al., 1996). linking unidentified membranous vesicles (Aunis and Bader, Whether β-catenin is required to stabilize the conformation of 1988; De Cesaris et al., 1989; Gregorio et al., 1993; Malchiodi- E-cadherin, as postulated (Chen et al., 1999), or whether β- Albedi et al., 1993; Zagon et al., 1986). These early findings catenin is needed to link E-cadherin to a spectrin scaffold in support a growing body of recent evidence indicating that, the early secretory pathway – as would be postulated by the beyond the Golgi, many if not all other compartments of the SAATS hypothesis (see below) – remains to be determined. secretory and endo/lysosomal pathways (Hoock et al., 1997; Also unanswered are the questions of whether spectrin actually Ziemnicka-Kotula et al., 1998) have associated spectrin binds to any part of the ER, and, if not, where in the skeletons. A major issue is now to understand whether the intermediate compartment the assembly of a spectrin coat is association of spectrin with the different intracellular initiated. compartments has any impact on the function of these At present, the evidence suggesting a role for spectrin in compartments. The evidence so far available indicates that this post-Golgi trafficking and in the control of endo/lysosomal is the case, at least for the Golgi-associated spectrin skeleton. compartments is limited. Specific isoforms of ankyrin (Hoock This evidence takes several forms. et al., 1997) and αΙ spectrin (Ziemnicka-Kotula et al., 1998) Along with the identification of targeting domains (MAD1 have been identified in endo/lysosomal compartments; spectrin and MAD2) in spectrin, it was observed that transfected βΙ and ankyrin associate with dynamin and clathrin in vitro and spectrin peptides encompassing region-I/MAD1 are potent in immunoprecipitates and fractionations of cell lysates (Cianci Spectrin in the secretory pathway 2339 et al., 1999a; Michaely et al., 1999), and the disruption of the pleomorphic vesicular tubular clusters (VTC) into ordered ankyrin-clathrin interaction inhibits clathrin-coated-pit Golgi cisternae would be sustained by the spectrin skeleton. In budding in vitro and endocytosis of fluorescent low density the later steps of the maturation-progression transport process, lipoprotein (LDL; Michaely et al., 1999). Finally, other dynamic control of the spectrin mesh would facilitate the components of the TGN and endocytic machinery, including release of Golgi membranes into post-Golgi compartments. annexin VI and amphiphysin, co-immunoprecipitate with βΙΙΙ Within this framework, agents that block association of spectrin (M. C. Stankewich, C. D. Cianci and J. S. Morrow, spectrin with Golgi membranes are envisioned to act by unpublished). impairing the organization and integration of incoming ER- derived membranes into the Golgi complex and thereby inhibit the transport of cargo molecules from the ER to the Golgi MODELS: THE GOLGI MESH AND THE SAATS (Devarajan et al., 1997a; Godi et al., 1998). HYPOTHESES To account for the role of spectrin in the movement of transport containers and proteins, and its apparent effects on Except for its possible inclusion as a Golgi-associated coiled- cargo selection in the secretory pathway, an alternative model coil protein (e.g. the golgin proteins, which share an overall envisages the presence of a generalized spectrin-ankyrin- organization reminiscent of spectrin, Kjer-Nielsen et al., 1999), adapter protein tethering system (SAATS; Fig. 4B; De Matteis spectrin does not belong to any previously recognized protein and Morrow, 1998; Devarajan et al., 1997a). This hypothetical class implicated in the management of traffic in the secretory model postulates that Golgi spectrin and its associated adapter pathway. Its pervasive presence in different compartments and proteins facilitate the sequestration of integral membrane the profound disturbances of transport that accompany its proteins into transport containers (or else the selective capture disruption suggest that it is central to the function of these of transport containers containing certain proteins) and compartments. Two related models have been proposed as facilitates their movement from the ER to the Golgi and working hypotheses (Fig. 4). beyond. SAATS probably involves adapter proteins other than One model emphasizes the role of spectrin as an organizing just ankyrin, and includes members of the protein 4.1, adducin, and stabilizing force in the Golgi (De Matteis and Morrow, α-catenin, β-catenin and actin-related protein families. Like 1998; Lorra and Huttner, 1999; Fig. 4A). The existence of a other coat complexes, SAATS assembly on nascent vesicles is Golgi scaffold or mesh, which by analogy with the plasma- regulated by ARF (Godi et al., 1998) and, presumably, SAATS membrane skeleton would act as a template to shape Golgi recognizes complex targeting signals resident in the membranes, is suggested by the presence of a residual cytoplasmic domains of integral membrane proteins, such as detergent insoluble Golgi-ghost structure containing spectrin, those that bind to ankyrin (see Fig. 3). In an alternative view, ankyrin, actin and other proteins (golgins) that are collectively SAATS can also be conceptualized as a kind of chaperone defined as a Golgi matrix (Beck et al., 1994; Fath et al., 1997; system sensitive to the fidelity of folding of the cytoplasmic Nakamura et al., 1995). Some components of the Golgi matrix domains of certain cargo proteins in the secretory pathway. In and a matrix-interacting protein, p115, have recently been either case, a key feature of the SAATS model is the role played shown to regulate the stacking process (Barr et al., 1998; by specific interactions between membrane proteins destined Shorter and Warren, 1999; Shorter et al., 1999). These proteins for anterograde transport and SAATS. When these interactions act by guiding the alignment and capture of adjacent cisternae occur, transport from the ER to the Golgi is efficient; when in a fashion analogous to the way that they tether transport they are blocked, transport is inefficient or blocked altogether. vesicles to donor/acceptor cisternae. Whether this tethering In contrast to the Golgi mesh hypothesis, in the SAATS mechanism per se is sufficient to shape the Golgi stacks, or model spectrin is envisioned to act first on the donor whether it acts in concert with a spectrin-based skeleton, compartment, clustering and selecting specific molecules or remains to be determined. Also undetermined is whether there cargo-laden vesicles that have already budded from the ER and are direct links between the recognized Golgi-matrix that are destined for anterograde-directed transport. This components and spectrin. Such an interaction of the Golgi compartment might be the ER itself, although it is more likely matrix with the ARF- and PtdIns(4,5)P2-sensitive spectrin that SAATS first acts on the VTCs of membranes emerging machinery might render the entire Golgi skeleton sensitive to from the ER in the intermediate compartment. This is a crucial G protein and phosphoinositide regulation, and explain the sorting station between anterograde-directed cargo molecules rapid and reversible Golgi destacking that accompanies and molecules targeted for retrograde delivery back to the ER inactivation of ARF by BFA. due to the presence of KK motif and phenylalanine residues in The Golgi scaffold/mesh model also fits well with recently their cytoplasmic domain. This is also the place where vesicles proposed versions of the cisternal progression-maturation shed their COPII coats and cluster prior to anterograde model (Bonfanti et al., 1998; Bannykh and Balch, 1997; transport along microtubule pathways (Bannykh et al., 1996; Mironov et al., 1997). An essential component of such a model Campbell and Schekman, 1997; Lippincott-Schwartz, 1998; is a dynamic Golgi scaffold able to undergo rapid remodeling. Presley et al., 1997; Rowe et al., 1996). It is at this point that The regulation of spectrin association with Golgi membranes SAATS is most likely to exert its effect, clustering a subset by ARF and the intrinsic expandability of a spectrin mesh (Fig. of the budded vesicles (those that contain sufficient 1) are both properties that would be important for capturing concentrations of protein or lipids that bind SAATS) into larger and remodeling of incoming pre-Golgi intermediates. The transport complexes that are then swiftly moved by spectrin- biochemical maturation of these elements into mature Golgi dynein/dynactin along microtubules to the cis-Golgi. Vesicles stacks would subsequently rely on the retrograde transport of in the intermediate compartment that are not captured by Golgi enzymes, whereas the morphological maturation of the SAATS, or that are extruded from the nascent clusters owing 2340 M. A. De Matteis and J. S. Morrow

Fig. 4. Models of the Golgi- associated Spectrin skeleton. (A) The Golgi spectrin mesh/scaffold. The mesh hypothesis attributes to spectrin a key role in organizing and stabilizing the Golgi membranes. In this model, spectrin is a crucial component of the Golgi scaffold, an array of proteins responsible for conferring shape and structural integrity on Golgi cisternae. Presumably, spectrin interacts with at least some of the Golgi matrix proteins. The spectrin-based scaffold is dynamic, undergoing cycles of assembly (top) or disassembly (bottom). These cycles, controlled by the small GTPase ARF and phosphoinositides, might be localized so as to permit budding or docking of transport vesicles, or more extended to allow the capture and remodeling of incoming pre-Golgi intermediates into ordered cis- Golgi cisterna and the stabilization of these cisternae as they mature into medial- and trans-cisternae. The dynamic and localized control of spectrin also facilitates the release of Golgi membranes into post-Golgi compartments at the trans pole. (B) The spectrin ankyrin adapter protein tethering system (SAATS). To explain the role of spectrin in both pre- and post-Golgi compartments, as well as the selective effect of spectrin disruption on cargo protein transport, the SAATS model attributes to spectrin a role as both a mechanism for cargo selection, as well as as a tethering link between transport containers and motors. COPII-laden vesicles bud from the ER and are released into the intermediate compartment (1). Although it is possible that the assembly of SAATS begins as early as the ER, perhaps in conjunction with the COPII-driven budding reaction (2), it is more likely that SAATS assembly begins in the intermediate compartment and is coincident with the shedding of the COPII coat from newly budded vesicles (1). A key feature of SAATS is its ability to bind selectively and cluster a subset of the budded vesicles (those that contain sufficient concentrations of protein or lipids that bind SAATS) into larger transport complexes that are then swiftly moved by dynein/dynactin along microtubules to the cis-Golgi (3). Vesicles in the intermediate compartment that are not captured by SAATS, or vesicles extruded from the nascent clusters owing to their lack of interaction with SAATS (and that are thereby depleted of proteins tethered by SAATS), presumably assemble COPI coats and are returned to the ER (1), or move anterograde but less efficiently than those facilitated by SAATS. In the Golgi (3), spectrin is envisioned to continue to function by organizing protein and lipid mosaics, thereby facilitating their retention and stabilization in the Golgi cisternae, as hypothesized for the Golgi mesh. As in the intermediate compartment, proteins not stabilized by SAATS might preferentially enter COPI-mediated retrograde pathways. At the TGN and beyond (4), spectrin-linked mosaics are sorted for delivery to downstream compartments in conjunction with other motor and budding systems. Spectrin in the secretory pathway 2341 to their lack of interaction with SAATS (e.g. see Fig. 2), between GRASP65 and GM130, components of a protein complex involved presumably assemble COPI coats and are returned to the ER, in the stacking of Golgi cisternae. EMBO J. 17, 3258-3268. or move anterograde but at much slower rates than those Beck, K. A., Buchanan, J. A., Malhotra, V. and Nelson, W. J. (1994). Golgi spectrin: identification of an erythroid beta-spectrin homolog associated facilitated by SAATS. with the Golgi complex. J. Cell Biol. 127, 707-723. Once assembled, it is also envisioned that SAATS might Beck, K. A. and Nelson, W. J. (1996). The spectrin-based membrane skeleton remain with its associated membrane mosaic as it is as a membrane protein-sorting machine. Am. J. Physiol. 270, C1263-1270. incorporated and transported through the Golgi; this process Beck, K. A., Buchanan, J. A. and Nelson, W. J. (1997). Golgi membrane skeleton: identification, localization and oligomerization of a 195 kDa probably would be accompanied by an exchange of at least ankyrin isoform associated with the Golgi complex. J. Cell Sci. 110, 1239- some SAATS components (e.g. the exchange of AnkG119 for 1249. the 195-kDa ankyrin). As vesicles bud from the TGN, SAATS Beck, K. A. and Nelson, W. J. (1998). A spectrin membrane skeleton of the might again play a role in directing a subset of cargo-laden Golgi complex. Biochim. Biophys. Acta 1404, 153-160. vesicles along microtubule pathways to the plasma membrane Berghs, S., Aggujaro, D., Dirkx, R., Zhang, J.-P. and Solimena, M. 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