Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2559-2563, April 1993 Review The molecular machinery for secretion is conserved from yeast to neurons Mark K. Bennett and Richard H. Scheller Howard Hughes Medical Institute, Beckman Center 8-155, Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305

ABSTRACT A variety of approaches involved in transport vesicle docking and family of involved in different have been utilized to identify and charac- fusion. Two approaches, the biochemical steps of vesicular transport. terize the molecules that mediate vesicular characterization of synaptic vesicles (the If this is the case, one might expect to trafficking along the secretory pathway. transport intermediate responsible for find VAMP-like proteins in simple eu- Two approaches that have been particu- the regulated release of karyotes, such as yeast. Indeed, several larly fruitful include the genetic dissection from neurons) and yeast genetics, have proteins have been identified in yeast that of the yeast secretory pathway and the provided results that suggest the mecha- have domain structures similar to, and biochemical characterization of proteins nisms involved in transport vesicle dock- with, VAMP. The involved in the membrane ing and fusion may be evolutionarily con- yeast most like VAMP is SNC1. trafficking in the mammalian nerve ter- served. The remarkable convergence of It shares 40% sequence identity with minal. The recent convergence of these these two approaches provides great VAMP (10). SNC1 was isolated by its approaches suggests that common mecha- ability to suppress loss of one of the promise for future analysis ofthe machin- functions of CAP (cyclase-associated nisms may underlie a wide variety of ves- ery involved in membrane trafficking. icle-mediated transport steps. We discuss protein). CAP is a 526-amino acid protein We will briefly discuss these results and component of the RAS-regulated adenyl- the results that support this possibility and propose a model for synaptic vesicle propose a model for synaptic vesicle dock- ate cyclase complex in yeast. The amino- docking and fusion that incorporates (i) terminal 168 residues of CAP are suffi- ing and fusion that incorporates evolution- common components that may be part of arily conserved elements that may be part cient for RAS to activate cyclase, the constitutive machinery involved in whereas the carboxyl-terminal 160 amino of a constitutive fusion machinery and many vesicular transport steps and a specialized elements that may mediate reg- (ii) acids of CAP are required for survival in set of regulatory components that may nutrient extremes. SNC1 suppresses ulatory events that are specific to the pro- confer calcium sensitivity to the specific only the loss of the carboxyl-terminal cess of neurotransmitter release. processes involved in neurotransmitter function of CAP and only in yeast ex- release. pressing an activated form of RAS2. Dis- Membrane trafficking along the exocytic The relative abundance of synaptic ruption of the SNC1 results in no and the endocytic pathways of eukary- vesicles in brain has made possible their apparent phenotype. However, a second otic cells is mediated by a series of ve- biochemical characterization. Nearly all VAMP homologue, SNC2, has been sicular intermediates. The fact that ve- synaptic vesicle proteins so far identified identified. Recent studies, including dis- sicular transport is vectorial suggests that are members of small families that ruption of both SNC , suggest that each step is mediated by specific target- are specific to neural or endocrine tissue. the SNC proteins are both localized to ing, docking, and fusion events. These One exception is the protein VAMP (ves- post-Golgi vesicles and required for events are likely to involve protein com- icle-associated ) (4-6) Golgi to plasma membrane trafficking (J. ponents localized to the transport vesicle or (7), an 18-kDa protein Gerst, personal communication). The re- and target membrane as well as soluble anchored to the cytoplasmic surface of lationship between the SNC proteins and factors. Although some components are the vesicle by a carboxyl-terminal trans- RAS is not clear at this time. expected to define the specificity of a membrane domain. VAMP It is clear however that part ofthe RAS particular transport step, others are Although is superfamily, members of the family likely to be common to multiple transport predominantly expressed in the nervous of GTP-binding proteins, is involved in steps. An additional level of complexity system, a protein at least immunologi- the regulation of vesicular trafficking in is involved in the regulated secretory cally related to VAMP has been identi- yeast and higher eukaryotes (11). The rab pathway, where the final step, exocyto- fied in adipocytes (8). In adipose cells, family is now known to contain >20 sis, is regulated by calcium or GTP. In the VAMP-like protein is associated with members. Many of these molecules are recent years, a combination of ap- vesicles that are responsible for the in- localized to specific intracellular mem- proaches, which includes yeast genetics, tracellular sequestration of the glucose brane compartments (12), including rab3 in vitro reconstitution of transport, and transporter GLUT4. In response to insu- on the synaptic vesicle (13). Biochemical biochemical characterization ofvesicular lin, these vesicles fuse with the plasma and genetic studies suggest that the rab intermediates, has led to the identifica- membrane, resulting in increased glucose family of low molecular weight GTP tion of a number of proteins that are transport into the cells. In Drosophila, binding proteins may mediate vectorial involved in membrane trafficking. For one form of VAMP is expressed predom- membrane trafficking. For example, example, NSF/secl8, a soluble protein inantly in the gut and malpighian tubules rabS, which is localized to the early en- required for membrane fusion at a num- (9). Whether nonneuronal VAMP func- dosome, regulates early endosome fusion ber of steps along the endocytic and tions in membrane trafficking remains to in vitro (14) and in vivo (15). In yeast, two exocytic pathways, was identified by be established. Although the extent of members ofthe rab family, YPT1 (16) and yeast genetics and in a mammalian in VAMP or VAMP-like protein expression vitro assay (1-3). outside the nervous system remains to be Abbreviations: VAMP, vesicle-associated In this review we will focus on several determined, these results raise the pos- membrane protein; CAP, cyclase-associated integral membrane proteins that may be sibility that VAMP is a member of a protein; ER, endoplasmic reticulum. 2559 Downloaded by guest on September 30, 2021 2560 Review: Bennett and Scheller Proc. Natl. Acad. Sci. USA 90 (1993) SEC4 (17), have been identified. YPT1 , has recently been identified in of higher eukaryotes. The yeast protein and SEC4 are required for vesicular the nerve terminal (22). Syntaxin, a 35- with the highest level of homology to transport from the endoplasmic reticu- kDa protein with a carboxyl-terminal syntaxin is an open reading frame near lum (ER) to the Golgi apparatus and from membrane anchor, was identified by its the 5' end of the gene for fatty acid the Golgi apparatus to the plasma mem- ability to interact with the synaptic ves- synthase (26). Genetic studies have re- brane, respectively. Several other pro- icle protein (p65). Anti- vealed that this open reading frame is one teins, involved in the regulation of vesic- bodies generated against syntaxin labeled of two genes, SSO and 5502, that are ular trafficking in the early secretory axonal membranes, particularly at syn- capable of acting as multicopy suppres- pathway in yeast, interact genetically aptic sites. These syntaxin antibodies sors of a temperature-sensitive allele of with YPT1. Two of these proteins, were capable of immunoprecipitating SEC1 (S. Keranen, personal communi- SLY12/BET1 (18, 19) and SLY2/SEC22 w-conotoxin binding sites, suggesting an cation). SEC1 is a hydrophilic protein (19, 20), have a domain structure similar interaction of syntaxin with neuronal required for proper transport from the to, and limited sequence homology with, voltage-gated calcium channels. Based Golgi to the plasma membrane (27). VAMP. on these properties it was proposed that SEC1 is itself a member of a family of These observations suggest that mem- syntaxin is involved in the docking of proteins involved in membrane traffick- bers of the rab and VAMP families are synaptic vesicles near voltage-gated cal- ing (28). SLY1 (19) and SLP1 (29), two involved in membrane trafficking at two cium channels in the presynaptic plasma proteins with sequence similarity to stages ofthe yeast secretory pathway and membrane. SEC1, are required for transport from the in the nerve terminal (Fig. 1 and Table 1). Recent studies of the yeast secretory ER to the Golgi and from the Golgi to the The colocalization of these protein fam- pathway have raised the possibility that vacuole, respectively. No mammalian ilies (SEC4 and SNC on post-Golgi ves- syntaxin is a member of a family of pro- homologues in the SEC1 family have yet icles, rab 3 and VAMP on synaptic ves- teins involved in vesicular trafficking. been described. icles) and their potential interaction (de- Three genes have been identified in yeast These findings suggest that members tected genetically for YPT1 and SLY12/ that encode proteins with a carboxyl- of several families of molecules (VAMP, SLY2 in ER to Golgi transport) suggest terminal membrane anchor and display- rab, syntaxin, and SEC1) may be re- that a rab protein interacting either di- ing significant homology to syntaxin, pri- quired for membrane transport in the rectly or indirectly with a VAMP-like marily over a 70-amino acid segment near nerve terminal and at several stages of protein may be components of a con- the membrane anchor. The first of these the secretory pathway in yeast (Fig. 1 and served machinery on the surface of ve- is SED5, a multicopy suppresser of loss Table 1). That the three syntaxin-like sicular transport intermediates that par- of ERD2 function (23). ERD2 is the ER yeast proteins are involved at distinct ticipates in docking or fusion. That retention receptor that is thought to rec- stages of the secretory pathway, coupled VAMP plays an important role in synap- ognize the amino acid sequence HDEL with the proposed role of syntaxin in tic vesicle docking or fusion is supported and function in retrieval of ER resident synaptic vesicle docking, raises the pos- by the recent observation that cleavage proteins from the Golgi (24). SED5 is sibility that these proteins function as of VAMP by the zinc-endoprotease as- itself required for a functional secretory target membrane-specific receptors for sociated with tetanus and botulinum pathway. Another yeast protein with ho- transport vesicles. Based on these results toxin is correlated with a block of neu- mology to syntaxin is PEP12 (25) (K. we suggest that members of membrane rotransmitter release (21). Becherer and E. Jones, GenBank se- protein families localized to the transport Components of the docking and fusion quence YSCPEP12P), which is required vesicle (VAMP and rab) and target mem- machinery localized to the target mem- for proper targeting of proteins to the brane (syntaxin) are components of the brane have been more difficult to iden- yeast vacuole, the functional equivalent molecular machinery that mediates spe- tify. A candidate vesicle docking protein, of the lysosome in the secretory pathway cifiic docking and fusion reactions in ER GOLGI PLASMA MEMBRANE

VACUOLE [

FIG. 1. Yeast proteins required at distinct positions in the secretory pathway. The members of four protein families involved in the yeast secretory pathway (syntaxin, VAMP, rab, and SEC1), and their proposed sites of action, are indicated. Downloaded by guest on September 30, 2021 Review: Bennett and Scheller Proc. Natl. Acad. Sci. USA 90 (1993) 2561

Table 1. Families of membrane proteins involved in the regulation of vesicular trafficking Family Yeast homologue and transport step Nerve terminal homologue rab YPT1 ER to Golgi rab3A SEC4 Golgi to plasma membrane VAMP BET1/SLY12 ER to Golgi VAMP1 or VAMP2 SEC22/SLY2 ER to Golgi SNC1 and SNC2 Golgi to plasma membrane Syntaxin SED5 ER to Golgi Syntaxin A or B PEP12 Golgi to vacuole SSO1 and SSO2* Golgi to plasma membrane *Open reading frame near FAS2. many vesicular trafficking processes. Al- tory elements likely to be specific to tion of synaptotagmin with syntaxin cou- though it is attractive to envisage a direct neurotransmitter release. The central el- pled with the interaction of syntaxin with or indirect connection between the two ements of the model are the rab, VAMP, voltage-gated calcium channels (22) place membranes involving these components, and syntaxin proteins, which, as dis- synaptotagmin in an ideal position to such a connection has not been demon- cussed above, may comprise a portion of respond to calcium. The observation that strated. It is ofinterest that as one moves a conserved constitutive docking or fu- catecholamine release from PC12 cells through the yeast secretory pathway to- sion machinery. However, the process of can be blocked by microinjection of ei- ward the plasma membrane, the vesicu- synaptic vesicle fusion is not constitutive ther antibodies against synaptotagmin or lar trafficking components become more but rather is tightly regulated by calcium. soluble fragments of synaptotagmin sup- homologous to the nerve terminal com- One candidate for the calcium-sensitive ports the possibility that this protein is an ponents. Thus VAMP is most like SNC, regulator is the synaptic vesicle protein important regulator of neurotransmitter rab3 is most like SEC4, and syntaxin is synaptotagmin (30, 31). Synaptotagmin is release (33). most like SSO. This suggests the inter- composed of an amino-terminal intrave- Another protein that interacts with esting possibility that the machinery re- sicular domain, a single transmembrane synaptotagmin is the a-latrotoxin recep- sponsible for secretion of neurotrans- domain, and a cytoplasmic domain made tor (34). a-Latrotoxin, a component of mitter is likely to have evolved directly up of two repeats homologous to the C2 black widow spider venom, binds to its from the trafficking machinery that me- regulatory domain of protein kinase C. plasma membrane-localized receptor and diates delivery of vesicles from the Golgi These C2-homologous domains have induces the efficient and calcium-inde- to the plasma membrane. been implicated in the calcium-depen- pendent fusion of synaptic vesicles with Our current model for synaptic vesicle dent interaction of synaptotagmin with the presynaptic plasma membrane (35). docking and fusion is presented in Fig. 2. membrane phospholipids (32). Thus, it The a-latrotoxin receptor is a member of This model incorporates the molecules has been proposed that synaptotagmin a family of proteins known as the neu- that have counterparts in the yeast secre- acts as a calcium sensor on the surface of rexins (36). The neurexins have a large tory pathway as well as potential regula- the synaptic vesicle. Indeed, the interac- extracellular domain, a single transmem-

.. . . . .---. . . VYNAPTInTAr.UIN_ v SYNAPTIC VAMP VESICLE MEMBRANE

RAB3

tetanus toxin

EII11 MEMBRANE SYNTAXIN a-latrotoxifl CALCIUM CHANNEL/

\NEUREXIN

FIG. 2. Proposed model for the molecular machinery involved in synaptic vesicle docking and fusion. Components that have conserved counterparts in yeast (syntaxin, VAMP, and rab3) form a constitutive machinery that is regulated by nerve terminal specific components (synaptotagmin, neurexin, and calcium channels). For a detailed discussion of the model, see the text. Interactions between the proteins (indicated by double-headed arrows) either have been detected in biochemical studies (syntaxin-synaptotagmin, syntaxin-calcium channel, and synaptotagmin-neurexin) or are inferred from genetic interactions in yeast (VAMP-rab3). The interactions may be either direct or mediated through additional molecules. The potential sites of action of a-latrotoxin and tetanus and botulimun toxins are indicated. Downloaded by guest on September 30, 2021 2562 Review: Bennett and Scheller Proc. Natl. Acad Sci. USA 90 (1993) brane domain, and a short intracellular reaction at other vesicular trafficking including interactions between synapto- region. At least three neurexin genes steps. In spite of extensive screening, no physin and VAMP and between synap- undergo extensive alternative RNA splic- synaptophysin homologues have yet totagmin and SV2 (59). It is possible that ing, resulting in a large number of iso- been identified in other systems. The such interactions contribute to the vesi- forms. The interaction between syn- generality of synaptophysin's proposed cle docking and fusion machinery. The aptotagmin and neurexins would be function awaits confirmation in other challenge for the future will be to estab- predicted to involve the cytoplasmic do- systems. lish the precise role of each protein, mains of the two proteins. We propose Another component of the synaptic whether as direct mediators or regulators that a neurexin, upon binding of a-latro- vesicle membrane is SV2 (42). cDNA of neurotransmitter release. The combi- toxin, induces a change in the conforma- clones encoding two forms of SV2 have nation of yeast genetics, biochemistry of tion of synaptotagmin or other proteins been identified (47-49). The SV2 proteins synaptic proteins, and in vitro reconsti- that mimics that induced by an influx of are composed of 12 membrane-spanning tution of vesicular transport should con- calcium. domains and display homology to bacte- tinue to provide insight into the con- The release of neurotransmitter from rial drug and nutrient transporters. It has served and unique components required the presynaptic nerve terminal occurs been suggested that these proteins play a for multiple membrane trafficking path- very rapidly following the influx of cal- role in the movement of molecules, such ways. cium (37). This suggests that the machin- as neurotransmitter, across the synaptic Many of the fundamental biochemical ery responsible for synaptic vesicle fu- vesicle membrane. However, the wide processes essential for living organisms sion may be preassembled or "primed" distribution of SV2 expression in the ner- are conserved across most species. En- in a prefusion complex. The conserved vous system suggests that its function is ergy metabolism, information flow components depicted in Fig. 2 may rep- not limited to a particular class of neuro- through DNA and RNA, and protein syn- resent a portion of such a complex. We transmitter. Another possible function thesis are examples of this conservation. suggest that the synaptotagmin/neurexin for a large protein with multiple mem- Thus, perhaps we should not be surprised complex may function as a negative reg- brane-spanning domains such as SV2 that homologues of molecules that medi- ulator (or inhibitor) of the constitutive might be in the process of membrane ate membrane flow in one ofthe simplest fusion machinery. This inhibition could fusion. As with synaptophysin, the role organisms, yeast, are critical compo- be relieved either by the influx ofcalcium of SV2, if any, in synaptic vesicle fusion nents of the most complex process ex- during nerve stimulation or by a-latro- remains to be established. hibited by living organisms, that of mam- toxin binding. PC12 cells selected for a The processes of synaptic vesicle malian brain function. We do not intend lack of synaptotagmin by complement- docking and fusion are potential targets to imply that when we understand mem- mediated cell killing are still capable of for the physiological modulation of syn- brane flow in yeast that we will com- regulated release of neurotransmitter aptic transmission. Such modulation of pletely understand the molecular basis of (38). This result is consistent with the synaptic efficacy is likely to contribute to synaptic transmission. Certainly all cel- idea that synaptotagmin is a negative such processes as learning and memory. lular processes have adapted a funda- regulator and that it is not absolutely A common mechanism by which protein mental set of biochemical principles to required for membrane fusion. If this is function can be modulated is protein suit specific evolutionary pressures. the case, an additional calcium-depen- phosphorylation. Several components of Since the conservation of molecules in- dent step would be required to explain the proposed synaptic vesicle docking volved in vesicular trafficking in yeast the observation that secretion from the and fusion complex are kinase sub- has proven useful for analyzing synaptic synaptotagmin-depleted PC12 cells is still strates. For example, in vitro syntaxin vesicle trafficking in the mammalian regulated rather than constitutive. and synaptotagmin are substrates for nerve terminal, so the regulatory mech- The final step in neurotransmitter se- casein kinase II (50). In addition, casein anisms that modulate protein transport in cretion, as in other vesicular transport kinase II appears to interact with synap- yeast may well provide clues as to mo- events, is membrane fusion. The earliest totagmin. Interestingly, casein kinase II lecular mechanisms by which higher or- detectable event in the fusion of biolog- activity increases in hippocampal slices ganisms modulate neurotransmitter re- ical membranes in other systems (viral following the induction of long-term po- lease for the purpose of learning and spike protein-mediated cell fusion and tentiation (51). A number of rab proteins memory. mast cell degranulation) is the formation have been shown to be phosphorylated in of a fusion pore (39). The molecular na- a cell cycle-dependent manner (52, 53), We thank the members of our laboratory for ture ofthis fusion pore, which may reflect suggesting that the function of these pro- many fruitful discussions. In addition, we a common fusion intermediate, remains teins might be regulated by phosphory- thank Dr. Jeffrey Gerst for discussions per- to be established. Several components of lation. Finally, synaptophysin is phos- taining to yeast SNC genes, Dr. Sirka Keranen the synaptic vesicle membrane may play phorylated on a tyrosine residue by for discussions pertaining to yeast 550 genes, a role in the fusion process. Synapto- pp6Osrc (54). The physiological conse- and Dr. Elizabeth Jones for discussion per- physin (40, 41), and the related synapto- quences of these phosphorylation events taining to the yeast PEP12 gene. Insight from porin (42), has four transmembrane do- have not been established. Dr. Hugh Pelham has been critical in putting mains and is present as a homooligomeric The multiple interactions among the together this manuscript. complex (43, 44). Upon reconstitution in synaptic vesicle and plasma membrane 1. Malhotra, V., Orci, L., Glick, B. S., artificial bilayers, synaptophysin can proteins illustrated in Fig. 2 may provide Block, M. R. & Rothman, J. E. (1988) form a transmembrane channel (45). It a scaffold upon which the complete ves- Cell 54, 221-227. has therefore been proposed that synap- icle fusion machinery is assembled. Ad- 2. Wilson, D. W., Wilcox, C. A., Flynn, tophysin, in conjunction with an as yet ditional components might include solu- G. C., Chen, E., Kuang, W. J., Henzel, undefined protein in the plasma mem- ble factors implicated in vesicle fusion W. J., Block, M. R., Ulrich, A. & Roth- brane with similar properties, forms a reactions such as NSF (55), SNAPs (sol- man, J. E. (1989) Nature (London) 339, pore across both bilayers (similar to a gap uble NSF attachment proteins; ref. 56), 355-359. junction) and that this pore represents the 3. Eakle, K. A., Bernstein, M. & Emr, annexins (57), and p145 (58) or membrane S. D. (1988) Mol. Cell. Biol. 8, 4098- first step in membrane fusion. If synap- proteins, both on the synaptic vesicle and 4109. tophysin functions in the formation of a on the plasma membrane. A number of 4. Trimble, W. S., Cowan, D. M. & fusion pore, other (related) proteins interactions among synaptic vesicle Scheller, R. H. (1988) Proc. Natl. Acad. might fulfill this function in the fusion membrane proteins have been detected, Sci. USA 85, 4538-4542. Downloaded by guest on September 30, 2021 Review: Bennett and ScheHer Proc. Natl. Acad. Sci. USA 90 (1993) 2563

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