Ikuron,

Vol. 6, 665-677, May, 1991, Copyright

0 1991 by Cell Press

Proteins of Synaptic Vesicles llnvolved in Exocytosis and Membrane Recycling Thomas C. Siidhof*

and Reinhard Jahn+

“Howard Hughes Medical Institute Department of Molecular Genetics [Jniversity of Texas Southwestern Medical Dallas, Texas 75235 tAbteilung Neurochemie Max-Planck-lnstitut fiir Psychiatric 8033 Planegg-Martinsried Germany

Synaptic Vesicles and Neurotransmitter

Center

Release

T-he past four years havewitnessed an explosion in our knowledge of the proteins that form small synaptic \,esicles. Molecular cloning of these proteins is rapidly leading to functional studies that define the specific roleof each of the proteins in the functions of synaptic \,esicles. In this article we review the recent progress i.1 this field, and we propose a classification of synaptic vesicle proteins based on their suggested functions. Synapses are abundant in brain. A single neuron can both receive and make thousands of synaptic contacts. The presynaptic nerve terminal is filled with small translucent synaptic vesicles. These are uniformly sized organelles of 40-50 nm diameter that store classic neurotransmitters such as glutamate, I-aminobutyric acid (GABA), and acetylcholine. Small synaptic vesicles account for approximately 7% of total brain protein (based on the abundance of synapsins la/b; these constitute 0.4% of total brain protein [Joelz et al., 19811 and 6% of total synaptic vesicle protein [Huttner et al., 19831). If synaptic vesicles were single molecules, their total concentration in brain would be micromolar. Many neurons secrete neuropeptides in addition to c lassie neurotransmitters. Neuropeptides are stored mostly in large dense-corevesicles in neurons and not i.1 the small translucent synaptic vesicles characterist c of synapses. Dense-core vesicles are less abundant tlan small synaptic vesicles in neurons and appear to contain a different set of proteins (reviewed in Stidt-of, 1989; DeCamilli and Jahn, 1990). Exocytosis of reuropeptide-containing vesicles is also Ca2+ depencent, but differs from that of the small synaptic vesicles in that it occurs outside of the synaptic active zone (Thureson-Klein, 1983; Pow and Morris, 1989). 7 hese observations suggest that neurons contain two independently regulated secretory pathways that use different protein components and are targeted to difft?rent intracellular sites. Because little is known about the structures of neuropeptide-containing vesicles, we will limit our discussion here to small translucent synaptic vesicles. A widely accepted view of the role of small synaptic vesicles in neurotransmitter release is as follows:

Review

When an action potential reaches the nerve terminal, the presynaptic plasma membrane depolarizes and voltage-gated Ca2+ channels open at the active zone. The ensuing rise in intracellular Ca2+ triggers exocytosis of synaptic vesicles, resulting in the release of neurotransmitters (Katz, 1966). The synaptic vesicle membranes are reclaimed from the plasma membrane byendocytosis,andthevesicleseventuallyrefiII with neurotransmitters. The cyclic pathway of synaptic vesicles can be divided arbitrarily into seven stages (numbered in Figure 1). After synaptic vesicles fill with neurotransmitters by active transport (stage I), they translocate to the active zone (stage II), where they dock at morphologically defined sites (stage Ill) of the presynaptic plasma membrane. Exocytosis of docked vesicles is triggered by Ca2+ with release of neurotransmitters (stage IV). Following exocytosis, the empty synaptic vesicles (devoid of neurotransmitters) form coated pits that rapidly undergo endocytosis (stage V) and move away from the active zone (stage VI). The clathrin coat is lost, and the vesicles become endocytotic vesicles. Synaptic vesicles then reform from the endocytotic vesicles, probably via a larger endosomal intermediate (stage VII). Synaptic vesicles have two fundamentally different functions: they take up and store neurotransmitters (stage I; Figure I), and they fuse with and bud from other membranes, most notably the presynaptic plasma membrane (stages II-VII). These functions must be performed by the proteins of synaptic vesicles, either alone or in concert with other components of the nerve terminal. The characterization of synaptic vesicle proteins constitutes a first step toward understanding the molecular mechanism underlying neurotransmitter release. With this aim in mind, we and others have studied the primary structures and biochemical properties of several synaptic vesicle proteins (Table 1). Indeed, at the present time the molecular description of the synaptic vesicle is more complete than that of most other organelles. The proteins of synaptic vesicles can be divided into two classes that correspond to the two functions of the vesicles. The first class consists of transport proteins, such as channels and pumps necessary for the uptake and storage of neurotransmitters. The second class consists of proteins involved in the targeted movement and fusion-fission reactions of the synaptic vesicle membrane. There are fewer synaptic vesicle proteins in the first transport class, and these are generally less abundant because only a few substances are transported across the synaptic vesicle membrane and not much transport activity is needed to fill the small intravesicular space. Most of the synaptic vesicle proteins that have been studied probably belong to the second class (Figure 2). Figure 1 depicts the cycle of synaptic vesicles in the nerve terminal as a single circular pathway, although

Figure 1. Pathway

of Synaptic

Vesicle

Movement

in the Nerve Terminal

Synaptic vesicles accumulate neurotransmitters by active transport (stage I) and then move to the plasma membrane (stage II), where they become docked at the active zone (stage Ill). Ca2+ influx after membrane depolarization triggers synaptic vesicle exocytosis and release of neurotransmitters (stage IV), after which the empty synaptic vesicles are endocytosed by clathrin-coated pits (stage V) and recycled (stage VI) via an endosomal intermediate (stage VII). Stages V and VII have not been definitively proven, but are made probable on the basis-of morphological observations (Heuser,-1989). -

this is probably an oversimplification. Synaptic vesicles are functionally heterogeneous. There appears to be an actively recycling population of synaptic vesicles and a population of “resting”vesicles that is utilized only upon extensive stimulation. Newly synthesized neurotransmitters are preferentially incorporated into the actively recycling vesicles (Barker et al., 1972) and are preferentially released upon stimulation. Several characteristics distinguish the synaptic vesicle pathway from other regulated secretory pathways. First, it is a local, autonomous pathway of the nerve terminal that is independent of organelles such as the Golgi complex or the trans-Golgi network. This contrasts with processes such as regulated secretion of peptide hormones that require recycling of secretory vesicles via the trans-Golgi network (Griffith and Simons, 1986). Second, synaptic vesicle exo- and endocytosis is much faster than that of other regulated pathways. Neurotransmitter release from a single nerve terminal can be triggered 50 times a second, requiring a coordinate and tightly linked regulation of the underlying biochemical processes (Steinbach and Stevens, 1976). Third, synaptic vesicles are stably

docked at the active zone before exocytosis. A secretory stimulus probably triggers exocytosis only of the vesicles that are already pre-docked (Almers and Tse, 1990). Synaptic vesicle release is highly regulated, not only in the coupling of exocytosis to membrane depolarization and Ca*+ influx, but also in the amount of neurotransmitter released per membrane depolarization. Recent studies showed that a variety of forms of longterm plasticity, including long-term potentiation in the hippocampus, enhance synaptic transmission by increasing neurotransmitter release (Malinow and Tsien, 1990; Bekkers and Stevens, 1990). There are two mechanisms by which neurotransmitter releasecould be enhanced: the number of active synaptic vesicles might be increased; or the probability that a single docked vesiclewill fuse might be increased. The latter seems to occur during long-term potentiation. It is likelythat this regulatory event is mediated by protein phosphorylation. However, its mechanism will not be understood until the proteins that function in the docking and fusion reactions of synaptic vesicles have been characterized.

Review: Synaptic Vesicle Proteins 667

Table 1. Proteins

of Synaptic

Name(s) Trafficking

Number of Amino Acids

Molecular Weight

Species

References

118 116 220 219 313 265 423 705 669 586 479 379

12,903 12,650 24,912 24,732 33,849 29,007 47,578 74,006 69,926 63,463 52,460 41,572 29,000 >100,000 86,000

Human Human Human Human Human Rat Human Human Human Rat Rat Torpedo Rat Torpedo Rat

Archer et al., 1990 Archer et al., 1990 Zahraoui et al., 1989 Zahraoui et al., 1989 iizcelik et al., 1990 Knaus et al., 1990 Perin et al., 1991a Stidhof, 1990 Siidhof. 1990 Sudhof et al., 1989c Stidhof et al., 1989c Linial et al., 1989 Baumert et al., 1990 Stadler and Dow, 1982 Buckley and Kelly, 1985

838

96,267 70 kd 56,661 31,495 38 kd 34 kd 33 kd 26,139 19 kd 15,849 30,061

Rat Bovine Human Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine

Perin et al., 1991c Stone et al., 1989 Stidhof et al., 1989b Wang et al., 1988 Stone et al., 1989 Stone et al., .I989 Stone et al., 1989 Hirsch et al., 1988 Stone et al., 1989 Mandel et al., 1988 Perin et al., 1988

Proteins

Synaptobrevin I (VAMP-I) Synaptobrevin II (VAMP-2) rab3A (SMC25A) rab3B (SMG25B) Synaptophysin Synaptoporin Synaptotagmin Synapsin la Synapsin lb ‘Synapsin Ila jynapsin Ilb JAT-1 J29 ‘roteoglycan (SVI) iv2 .-ransport

Vesicles

Proteins

‘/acuolar proton pump’ 116 kd subunit 70 kd subunit 58 kd subunit 40 kd polypeptide 38 kd polypeptide 34 kd polypeptide 33 kd polypeptide 31 kd polypeptide 19 kd polypeptide 17 kd polypeptide (Iytochrome b561

513 273

226 155 273

LVhenever possible, the human structures are cited. Molecular mass values are given (in kilodaltons) as calculated from the cDNA sequence. Where such data are not available, apparent molecular weights are given. In addition to those proteins listed here, the f,,llowing proteins have been functionally identified: ceramide kinase (Bajjalieh et al., 1989); GABA, glutamate, catecholamine, and acetylcholine transporters (reviewed in Maycox et al., 1990); a src-related tyrosine kinase (Pang et al., 1988, Sot. Neurosci., abstract); and Ca2’/calmodulin-dependent protein kinase II fJahn and Siidhof, unpublished data). d Not all subunits listed were from brain, and not all were definitely shown to be subunits of the proton pump. It is possible that there are differences between brain and non-brain subunits of the proton pump. ___-___

In the following sections, we discuss the evidence implicating some of the well-characterized synaptic vesicle proteins in specific stages of the synaptic vesic!e pathway. The emphasis will be on the most abundant proteins of synaptic vesicles (some of which are sl:hematically depicted in Figure 2) for which defined functions have been suggested.

Neurotransmitter

Uptake and Transport Proteins

Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient across the synaptic vesicle membrane. The gradient is generated by a proton pump in the synaptic vesicle membrane that belongs to a family of vacuolar proton pumps and is probably identical to the wellcharacterized proton pump of brain clathrin-coated vesicles (reviewed in Stone et al., 1989). It is likely that the same pump recycles between synaptic vesicles, coated pits, and endocytotic vesicles (Figure 1). The biochemical properties of all these proton pumps are

similar, and more than 90% of the vacuolar proton pump in brain copurifies with synaptic vesicle markers (Jahn and Stidhof, unpublished data). The vacuolar proton pump is a large heterooligomeric complex, containing eight to nine different subunits, most of which have been cloned (Table 1). The catalytic ATPase domain and the transmembrane proton channel of the vacuolar pump resemble those of the mitochondrial and bacterial proton ATPases. However, no similarity could be detected between the other subunits of the vacuolar pump and those of other proton ATPases. In addition to synaptic vesicles, vacuolar proton pumps are found in other organelles, particularly lysosomes and endosomes. Functional differences have been reported among pumps of different organelles, but it is not known whether these are caused by differences in subunit composition or subunit structure. One of the subunits, the58 kd protein, is expressed in different forms in brain and kidney(Bernasconi et al., 1990; Sudhof et al., 198913). Another subunit (116 kd) is alternatively spliced (Perin et al., 1991c). These observations sug-

Figure 2. Structures

of the Major

Trafficking

Proteins

of Synaptic

Vesicles

That Have Been Molecularly

Characterized

The proteins are drawn approximately to scale, although the synaptic vesicle in which they reside is drawn proportionally smaller. All of the proteins shown here have putative functions in the trafficking of synaptic vesicles in the nerve terminal. Most of the major synaptic vesicle proteins exist in multiple isoforms (Table I), which are not necessarily colocalized on a single synaptic vesicle.

gest that different isoforms of the proton pump may be localized in different organelles. Synaptic vesicles contain specific neurotransmitter transporters that are energetically coupled to the electrochemical protongradient.Thevesicular neurotransmitter transporters are structurally and mechanistically different from the Na+-dependent neurotransmitter transporters localized to the presynaptic plasma membrane. Several of the vesicular transporters have been solubilized and reconstituted into proteoliposomes (reviewed in Johnson, 1988; Maycox et al., 1990), and a monoamine carrier that is probably identical to that of synaptic vesicles has been purified from chromaffin granules (Stern-Bach et al., 1990). So far, transporters have been demonstrated for catecholamines, acetylcholine, glutamate, and GABA. It appears that all catecholamines, serotonin, and histamine utilize the same transporter. CABA and glycine also seem to share one transporter (Hell and Jahn, unpublished data). In addition, only one transporter for excitatory transmitters (strongly glutamate preferring) has been discovered. Therefore the number of different neurotransmitter transporters in synaptic vesicles may be quite low, possibly as few as four,

which is in striking contrast to the large number of different neurotransmitter receptors or plasma membrane transporters that have been described. Thetransport mechanismsforthe individual neurotransmitter transporters in synaptic vesicles vary. Uptake of different neurotransmitters is differentially coupled to the membrane potential or the proton gradient component of the electrochemical gradient (Maycoxetal.,1990). Sincetheproton pump iselectrogenie, the generation of a pH gradient is dependent on charge-balancing counter-ions, which are provided in Cl-via influx through a channel in the vesicle membrane. Such a channel has been purified from bovine brain clathrin-coated vesicles (Xie et al., 1989), but has not been structurally identified in synaptic vesicles. Synapsins: Navigating the Cytoskeleton

Synaptic

Vesicles

along

Synaptic vesicles move to the active zone in preparation for exocytosis and move away from it after endocytosis (stages II and VI; Figure 1). Quick-freeze deepetch electron microscopy demonstrated that the

Ceview: Synaptic Vesicle Proteins f69

COMMON

I 5 ynapsm

PP I

la B

E PPD

Synapstn

lb

A P

B

A

B

P I

PROLINE B

VA

C I

A P

I

RICH

I

5ynapsin

P 1

PROLINE A P

Figure 3. Domatn

VARIABLE

I

I

RICH

jlfl

C I

D

Model

of the Synapsins

The four synapsins are drawn to scale, with the names of the svnaosins shown on the left. Putative protein domains are labeled below the respective domains with the letters A-l. Identified phosphorylation sites are shown above the protein bar bv the letter P. (Figure modified from Stidhof et al., 1989c.I

F

C lG

H

lib C

G I

nerve terminal is filled with a meshwork of microfilamerits and microtubules (Landis et al., 1988; Hirokawa e; al., 1989). Synaptic vesicles in the nerve terminal must navigate around this cytoskeleton, which would o’:herwise serve as an impenetrable barrier. This navigation may be mediated by synapsins, a family of abundant peripheral membrane proteins that were dlscovered by Paul Creengard’s laboratory as the majc’r phosphoproteins in nerve terminals (Johnson et al., 1972). Synapsins are specific for synaptic vesicles in neurons and bind to several elements of the cytoskeleton, suggesting a general role in the guidance oi synaptic vesicles to their release sites (DeCamilli et al., 1983; Baines and Bennett, 1985,1986; Petrucci and Morrow, 1987; Bahler and Creengard, 1987; Sudhof et al ., 1989c). Structure and Distribution The synapsin family consists of four homologous proteins-synapsins la, lb, lla,and IIb.ThemRNAsencoding the four synapsins are derived by differential splicing from the primarytranscripts of two genes (Sudhof et al., 1989c; Siidhof, 1990). Differential splicing of both the synapsin I and the synapsin II RNA occurs in the carboxy-terminal half of the coding region, resulting in two pairs of proteins with identical aminoterminal and divergent carboxy-terminal sequences (synapsins la and lb [la/b] and synapsins Ila and Ilb [Ila/b], respectively). Comparison of the amino acid sequences of synapsins la/b with those of synapsins Ila/b reveals a high degree of homology, starting at the amino terminus and extending over more than 400 amino acids. On the basis of this homology and of sequence characteristics, a domain model for the four synapsins can be advanced (Figure 3). According to this model, the four synapsins share a set of homologous amino-terminal domains (domains A-C) and diverge at different points from each other in their carboxy-terminal domains. The carboxyl termini consist of sets of differentially spliced domains that are different among all four synapsins (domains E, F, H, and I). Interestingly, how-

ever, the final domains of synapsins la and Ila are again highly homologous (domain E), although no similarity exists between the other two synapsins in the final domain. The central homologous region of the synapsins (domain C) is separated from the differentially spliced carboxy-terminal sequences (domains E, F, H, and I) by additional domains that are weakly homologous to each other in synapsins la/b and llaib (domains D and G, respectively). Domain D is much larger than domain C and contains phosphorylation sites for several protein kinases that functionally affect synapsins la/b (see below). Domain D has an unusual amino acid composition in which proline accounts for 27% and glutamine for 17% of the total residues, whereas no asparagine or aromatic amino acids are found. Domain D contains 17% of its residues as positively charged amino acids but less than 1% as negative charges, creating a charge imbalance that may confer onto synapsins la/b their unusual biochemical properties. The four synapsins are differentially distributed in brain. Some synapses contain all four synapsins, whereas others lack one or two isoforms (Sudhof et al., 1989c). Synapsin lb is the most abundant form and is present in virtually all synapses. Not all four synapsins are needed for a synapse to be functional, and the expression of synapsin isoforms can be regulated between neurons. Since the regulation of the expression of synapsin isoforms operates at the level of both transcription (synapsins la/b versus Ila/b) and alternative splicing (synapsins la versus lb and Ila versus [lb), this regulation involves two different longterm mechanisms with different time constants. Phosphorylation The four synapsins were originally discovered as major substrates of endogenous CAMP-dependent protein kinases in mammalian brain (Johnson et al., 1972). In-depth studies demonstrated that synapsins la/b contain at least four distinct phosphorylation sites that are substrates for four different protein kinases (Huttner et al., 1981; Kennedy and Greengard, 1981;

Neuron 670

Hall et al., 1990). In contrast, synapsins Ila/b exhibit only a single site that is phosphorylated by two different protein kinases (Huang et al., 1982). All synapsins contain the same amino-terminal phosphorylation site for CAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase I (domain A; Figure 3) (Czernik et al., 1987; Sudhof et al., 1989c). In the carboxy-terminal domain D of synapsins la/b, which is absent from synapsins Ilalb, two different phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II and a separate site for proline-directed protein kinase are found (Czernik et al., 1987; Hall et al., 1990). Although the functional significance of most of the phosphorylation sites in the synapsins is not yet known, it seems likely that phosphorylation serves as a regulatory signal. This is strongly supported by the tight regulation of the phosphorylation of synapsins la/b in vivo (reviewed in Nestler and Greengard, 1984). Synapsins la/b show marked conformational changes as a function of phosphorylation (Benfenati et al., 1990). Phosphorylation of these synapsins at their carboxy-terminal sites byCa*+/calmodulin-dependent protein kinase II modulates the binding of these synapsins to synaptic vesicles (Schiebler et al., 1986) and to actin (BPhler and Greengard, 1987; Petrucci and Morrow, 1987). Binding to Cytoskeletal Proteins In vitro synapsins la/b bind to several elements of the cytoskeleton, including microfilaments, microtubules, and spectrin (Baines and Bennett, 1985, 1986; Bahler and Greengard, 1987; Petrucci and Morrow, 1987). Synapsins la/b not only bind to actin filaments, but also bundle them, suggesting the presence of several actin-binding sites. Actin filament bundling, but not binding, by synapsins la/b is inhibited by phosphorylation at the carboxy-terminal Ca2+/calmodulindependent protein kinase II site. Chemical cleavage of synapsins la/b localizes at least one actin-binding site to the central homologous region of the protein (Petrucci et al., 1988; Bihler et al., 1989). Since this region is highly conserved between synapsins la/b and synapsins Ila/b, it is likely that synapsins Ila/b also bind actin (Sudhof et al., 1989c). The absence of sequence homology between the synapsins and other actin- or microtubule-binding proteins suggests that the synapsins constitute a novel family of actinbinding proteins (Sudhof et al., 1989c). Functions Based on the in vitro interactions of synapsins with actin microfilaments and other elements of the cytoskeleton, it was proposed that synapsins mediate the interactions of synaptic vesicles with the cytoskeleton in vivo (Bahler and Creengard, 1987; Steiner et al., 1987). This model predicts that synapsins would regulate neurotransmitter release by determining the availability of synaptic vesicles for exocytosis. The model is supported by the following evidence:

-Quick-freeze, deep-etch electron microscopy revealed that fine filaments link synaptic vesicles to the cytoskeleton of the nerve terminal. These filaments were identified as synapsins la/b, which were suggested to bind to the vesicles by their carboxyterminal tails and to microfilaments by their aminoterminal heads (Landis et al., 1988; Hirokawa et al., 1989). However, the assignment of the head and tail domains of synapsins la/b in these studies does not fit well with the known functional domains of the synapsins, and synapsins llalb were not considered in this model. -Depolarization of synaptosomes induces rapid actin polymerization, which correlates with neurotransmitter release and is followed by immediate depolymerization (Bernstein and Bamburg, 1989). Under similar conditions, a portion of synapsins la/b was released from synaptic vesicles in synaptosomes, suggesting a link between synapsin localization and actin assembly (Sihra et al., 1989). - Phosphorylated and dephosphorylated mammalian synapsins la/b were injected into the preterminal digit of the squid giant axon (Llinas et al., 1985). Dephosphorylated synapsins la/b, but not phosphorylated synapsins la/b, strongly inhibited neurotransmission, correlating with the effect of phosphorylation on the actin-bundling properties of synapsins la/b (Petrucci and Morrow, 1987; BPhler and Greengard, 1987). It is currently not known whether synapsins or homologs of synapsins are present in invertebrates such as squid. -Introduction of Ca2+/calmodulin-dependent protein kinase II into the squid giant synapse or into synaptosomes induced an increase in the depolarization-induced release of neurotransmitters (Llinas et al., 1985; Nichols et al., 1990). However, it is unclear whether the introduced kinases exerted their stimulatory effects on synapsins la/b. Previous experiments had demonstrated that the synapsins are already stoichiometrically phosphorylated under these conditions bythe endogenous kinase in mammalian synaptosomes (reviewed in Nestler and Creegard, 1984). In summary, the localization and abundance of the synapsins are consistent with a function in the synaptic vesicle pathway and in the targeting of vesicles destined for neurotransmitter release. The regulation of the expression of different forms of synapsins by transcription and alternative splicing and the extensive phosphorylation of synapsins by different protein kinases suggest that this role may be subject to both long- and short-term regulation. The avid and specific interactions of synapsins with synaptic vesicles and different elements of the cytoskeleton suggest that their function may relate to the movement of synaptic vesicles to and from the active zone before and after exocytosis. However, the specifics of this function remain to be established. It is likely that synapsins do not simply regulatethe binding of synaptic vesicles to microfilaments because their multifaceted biochemical properties suggest additional roles and because

7eview: 171

Synaptic

Vesicle

onlyactin bundling, to phosphorylation.

Proteins

and not actin binding,

I ab3A: Targeting Synaptic to the Active Zone

is sensitive

Vesicles

‘synaptic vesicles are specifically targeted to the active zone, where they dock and exocytose. The docking of synaptic vesicles to the presynaptic plasma membrane results in a stable complex that is confined to the active zone (stage III in the synaptic vesicle pathway; Figure 1). Small GTP-binding proteins (G prot.:ins) of the ~21” superfamily have been suggested te> mediate the precise recognition of interacting membrane compartments preceding membrane fusion (Bourne, 1988). In yeast, mutations in sec4, agene encoding a small G protein, prevent docking and fuslon of exocytotic secretory vesicles (Salminen and Novick, 1987). By analogy, a small G protein may be required for the docking and fusion of synaptic vesicres. Since only synaptic vesicles dock at the active z3ne, this small G protein should be specific for synaptic vesicles. Synaptic vesicles do in fact contain a small C protein named rab3A that is specific for neuronal vesicles (Fischer v. Mollard et al., 1990a). A c’osely related homolog named rab3B has also been characterized (Matsui et al., 1988; Zahraoui et al., 1’489). rab3A is present in two pools in neurons: 70%-80% oi rab3A is tightly bound to synaptic vesicles and can be removed only by detergents, and 20%-30% of rab3A is soluble (Fischer v. Mollard et al., 1990a). The soluble and membrane-bound forms of rab3A both contain a hydrophobic domain that is absent from bacterially produced rab3A, suggesting the presence of a posttranslational modification. The hydrophobic group of soluble rab3A is hydroxylamine sensitive, indicating that it is ester linked, whereas that of the membrane-bound form is not. This indicates that rab3A may contain two different hydrophobic modifications: one is probably ester linked and may represent palmitate, and the other is hydroxylamine resistavlt and is present only on the membrane-bound form. It is likely that the latter causes the tight association of rab3A with the synaptic vesicle membrane. rao3A contains a conserved Cys-X-Cys sequence at its carboxyl terminus, which differs from the Cys-A-A-X sequence that is used for isoprenylation (Hancock et al., 1989). This sequence may bethe site for someother posttranslational modification of rab3A; however, rab3A also appears to be polyisoprenylated (Johnston et al., 1991). Sased largely on the work on sec4 in yeast, a model was proposed to explain the mechanism of action of small G proteins in membrane recognition and fusion-fission events (Bourne, 1988; Walworth et al., 1989). Before the membranes interact, the G protein is Dound to the vesicles in its GTP-containing form. Dl.ring membrane contact, the G protein binds to a specific receptor molecule in the target membrane.

This interaction is thought to be essential to ensure that only the proper membrane compartments bind, resulting in a prefusion complex. Then fusion occurs, followed byendocytotic membrane retrieval (fission). GTP hydrolysis before or after the fusion step accounts forthe unidirectionalityof the membrane flow and is triggered by the interaction of the G protein with a GTPase-activating protein. At some stage after fusion, the GDP-bound form of the small G protein dissociates from the membrane, possibly in response to removal of its hydrophobic modification that binds it to the membrane. The G protein binds anew to the vesicles only after its GDP has been exchanged for GTP. This model predicts that fusion-fission events should involve cycling of small G proteins between membranebound and free states. Furthermore, the cycles of the small G proteins between bound and free states should be associated with two parallel metabolic cycles: GTP hydrolysis and GDP-GTP exchange, as well as removal and attachment of a hydrophobic modification. Some of the predictions made by this model can be tested in nerve terminals more directly than in many other systems. In this regard, it was recently shown that rab3A reversibly dissociates from the synaptic vesicle membrane after exocytosis in synaptosomes (Fischer v. Mollard et al., 1990b). This observation suggests a function of rab3A in the docking or fusion of synaptic vesicles and is the first direct evidence for an association-dissociation cycle of a small G protein. A protein named GDP dissociation inhibitor has been purified from bovine brain. This protein inhibits the exchange of GTP for GDP in rab3A (Sasaki et al., 1990) and catalyzes the removal of the GDP-bound form of rab3A from the membrane (Araki et al., 1990). These findings are consistent with the hypothesis that membrane removal of rab3A is triggered by GTP hydrolysis. Most of the proteins interacting with rab3A during its cycle remain to be characterized, in particular the docking receptor and the GTPase-activating protein in the active zone (which may or may not be identical). Furthermore, additional G proteins may be involved with similar cycles operating on different stages of the synaptic vesicle pathway, for example, in the fusion of endocytotic membranes with endosomes. Synaptotagmin:

Docking

and Fusing Synaptic

Vesicles

As discussed above, synaptic vesicles are docked to the presynaptic plasma membrane before they undergo exocytosis. Freeze-fracture electron microscopy of the presynaptic plasma membrane containing bound synaptic vesicles demonstrated the presence of large intramembraneous particles of three to five subunits (Pfenninger et al., 1972). These particles may represent membrane proteins involved in synaptic vesicle docking and fusion. The proteins directing docking and fusion of synaptic vesicles are unknown. However, they must exhibit certain defined properties. First, there must be pro-

NWNXl 672

teins capable of forming an oligomeric protein complex with relatively large subunits. Such a complex may represent the core of the synaptic vesicle-plasma membrane attachment point as revealed by freezefracture. Second, a Ca2+ receptor (not necessarily of high affinity) must be part of the complex to trigger fusion. Third, regulated interaction of a “fusion protein” with the phospholipid bilayers of the participating membranes must occur, allowing the rearrangement of phospholipids during fusion. There are two candidate proteins in synaptic vesicles that fulfill some of these requirements: synaptotagmin and synaptophysin. Synaptotagmin (M, 65,000) is an abundant integral membrane protein of synaptic vesicles (Matthew et al., 1981; Perin et al., 1990,1991a, 1991b). Although it lacks a cleaved signal sequence, synaptotagmin contains a small amino-terminal intravesicular domain that is glycosylated. This is followed by a single transmembrane region and a large carboxy-terminal cytoplasmic sequence that contains two copies of an internal repeat. These repeats are homologous to the CL domain in the regulatory region of protein kinase C. The two C2 domain repeats in synaptotagmin are only slightly more homologous to each other than they are to thecorresponding region in protein kinaseC(Perin et al., 1990, 1991a, and 1991b). Synaptotagmin, like rab3 and synaptobrevin, also appears to be present in multiple isoforms in rat brain (Geppertand Sudhoff, unpublished data). In addition, a homolog of synaptotagmin has been cloned from Drosophila melanogaster (Perin et al., 1991a). Its sequence demonstrates that the cytoplasmic domains of synaptotagmin are evolutionarily highly conserved, with the highest degree of conservation in the CZ domain repeats (Perin et al., 1991b). The differences between the two consecutive C2:domain repeats are well conserved, suggesting a functional differentiation of the two synaptotagmin repeats. Two classes of protein kinase C isoenzymes have been described based on whether they contain or lack a CZ domain. Functionally, these two classes show a single consistent difference: those that contain the regulatory C2 domain require Ca*’ for activation, where as those that lack it are Ca*+ independent (reviewed in Nishizuka, 1989). Thus, the C2 domain in protein kinase C may be a Ca*+-binding domain that causes protein kinase C to be translocated to membranesand to be regulated by Ca *+. It is likely that the CZ domains of synaptotagmin perform a similar role. To date, however, no evidence for Ca*+ binding to the C2 domains of synaptotagmin has been obtained (Perin et al., 1990). If this binding is of low affinity, it would be difficult to demonstrate. Surprisingly, a high affinity phospholipid-binding activity was found associated with the cytoplasmic domain of synaptotagmin produced as recombinant protein in bacteria (Perin et al., 1990,199la). Phospholipid binding was conserved between human, rat, and Drosophila synaptotagrnins and was restricted to

phospholipids that contain negatively charged head groups and two acyl chains. This specificity suggests that synaptotagmin does not indiscriminately recognize negatively charged surfaces, but also interacts with the hydrophobic interior of the bilayer. The phospholipid-binding properties of synaptotagmin allow it to agglutinate red blood cells, which contain negatively charged phospholipids on their surface. However, a recombinant synaptotagmin construct that contained only a single C2 domain was unable to hemagglutinate, although it still bound radiolabeled phosphotidylserine (Perin et al., 1991a). This indicates that both C2 domains may be capable of phospholipid binding. It will be interesting to determine whether the CZ domain in protein kinase C also constitutes a phospholipid-binding domain. Native rat brain synaptotagmin is present in dimers that are organized in high molecular weight complexes of approximately 240 kd (Perin et al., 1991b). It is currently unknown whether these complexes are composed of two synaptotagmin dimersor one synaptotagmin dimer plus other proteins. In addition to phospholipid binding, synaptotagmin was reported to bind calmodulin (Trifaro et al., 1989). However, its affinity for calmodulin is very low (Perin et al., unpublished data), suggesting that this binding site is not physiologically relevant. In summary, synaptotagmin exhibits several properties that would be desirable of a protein involved in synaptic vesicle docking and fusion: it may have one or two Ca*“-binding sites per monomer; it interacts avidlywith phospholipidsthatare present on the interior surface of the plasma membrane; and it is part of a high molecularweight complexthat contains several copies of synaptotagmin, which should potentiate its binding activities. Synaptophysin: Pore Complex

A Candidate for the Exocytotic

Synaptophysin is probably the most abundant integral membrane protein of synaptic vesicles (Jahn et al., 1985; Wiedenmann and Franke, 1985). It contains four transmembrane regions and a cytoplasmic carboxy1 terminus consisting of ten copies of an imperfect tyrosine-rich repeat that is probably phosphorylated (Sudhof et al., 1987; Leube et al., 1987; Pang et al., 1988). Native synaptophysin in synaptic vesicles consists of a high molecular weight complex that appears to contain three to four subunits of synaptophysin (Rehm et al., 1986) and a second, unidentified low molecular weight protein (Johnston and Sudhof, 1990). Initial studies suggested that synaptophysin may bind Ca2+ (Rehm et al., 1986). However, this finding could not be confirmed in later studies (Johnston et al., unpublished data). Purified synaptophysin was shown to form voltagedependent channels in black lipid membranes (Thomas et al., 1988). It was postulated that synaptophysin is a channel protein that is structurally and functionally

Review: Synaptic Vesicle Proteins 673

similar tothegap junction protein connexin. Connexins form pores between adjacent cells, allowing the passage of small molecules. In addition, it was proposed that synaptophysin may correspond to cationselective channels that were observed by patch clamping in fused synaptic vesicles from Torpedo marmorata (Rahamimoff et al., 1988). However, these conclusions may not be completely accurate for the following reasons: -The purified synaptophysin used for the channel measurements in the black lipid membranes (Thomas et al., 1988) was quite different from native synaptophysin (Johnston et al., 1989a; Johnston and Sudhof, 1990). It probably contained rearranged disulfide bonds, had lost its low molecular weight protein subunit, and was aggregated into larger complexes than native synaptophysin, which does not form hexamers (Rehm et al., 1986; Johnston and Sudhof, 1990). -Connexins and synaptophysin both contain cytoplasmic amino and carboxyl termini and four transmembrane regions. Therefore, they are structurally similar without exhibiting sequence similarity. How‘ever, gap junctions are formed by interactions between the extracellular loops of the connexins from two different cells. If synaptophysin in synaptic vesi.Aes formed an analogous pore with a presynaptic olasma membrane component, this would be possiale only via its cytoplasmic sequences. Therefore oores formed by connexins and synaptophysins would “lave opposite orientations. -The channels observed in black lipid membranes .after incorporation of purified synaptophysin were zinearly activated by positive voltages (Thomas et al., 1988). Synaptic vesicles have an inside-positive memJrane potential that is probably higherthan thatof the !alasma membrane(Maycoxet al., 1988). Sincedocking .ind fusion result in a reduction of the potential differe?nce across the vesicle membrane, the synaptophysin 1:hannels observed would beopen primarily in resting vesicles and would partially close upon docking of the vesicles. It seems likely that native synaptophysin in free syn.iptic vesicles does not form a channel and that its structure is quite different from that of the connexins. .nstead of this model, we would like to propose that :;ynaptophysin may participate in the formation of the putative fusion pore during synaptic vesicle exocytosis by forming an unstable pore which differs prom that of other channels. One of the most striking properties of synaptophysin is its tendency to form homo-oligomers when it is released from the conc;traints imposed upon it by the phospholipid bilayer and its labile intramolecular disulfide bonds (John!>ton and Siidhof, 1990). It is quite possible that the channels observed with reconstituted purified synaptophysin represent the artifactual equivalent of an activated state. If so, native synaptophysin should normally reach this activated state only by its interaction with a presynaptic plasma membrane protein. Indeed, a recent report from Heinrich Betz’s laboratory

suggest that such a protein can be isolated (Thomas and Betz, 1990). It is also interesting that synaptophysin, when expressed in transfected fibroblasts, is sorted into recycling microvesicles that colocalize with the receptor-mediated endocytotic pathway (Johnston et al., 1989b). The sorting of synaptophysin in these cells is so efficient that most of the microvesicles contain synaptophysin as their major protein component (Leube et al., 1989; Johnston et al., 1989b). These findings support an intrinsic relationship between the synaptic vesicle pathway and the receptor-mediated endocytotic pathway, both of which utilize clathrincoated intermediates (see below). They also point toward a potential role of synaptophysin in organizing these vesicles into stable organelles.

Endocytosis and Recycling of Synaptic Vesicles Synaptic vesicle antigens are quickly retrieved from the presynaptic plasma membrane after exocytosis (stage V; Figure 1) and probably recycle by going through an endosomal intermediate (stages VI and VII). The existence of an endosomal intermediate is a conjecture based on the appearance of internal spaces labeled with endocytotic markers after synaptic vesicle endocytosis (Heuser and Reese, 1973). The endocytosis of synaptic vesicles appears to occur by clathrin-coated pits, as suggested bythefollowing evidence: -Morphologically, coated pits are observed at the synapse by quick-freeze experiments immediately after stimulation of the neuromuscular junction (reviewed in Heuser, 1989). -In the temperature-sensitive Drosophila mutant shihire, neuromuscular paralysis, caused by a failure of exocytosed synaptic vesicles to re-endocytose is observed at the restricted temperature (Salkoff and Kelly, 1978). It was shown that this phenotype represents a general endocytotic defect that is also observed for receptor-mediated endocytosis outside of the nervous system (Kosaka and Ikeda, 1983). This demonstrates a common mechanism for endocytosis of synaptic vesicles and receptor-mediated endocytosis. -Clathrin-coated vesicles and synaptic vesicles isolated from brain show a considerable overlap in their constituent proteins, suggesting that they represent different branches of the same pathway (Pfeffer and Kelly, 1985; Maycox, Sudhof, Perin, Morris, Ungewickell, and Jahn, unpublished data). It is possible that other endocytotic pathways are also being used for synaptic vesicle membrane retrieval in addition to clathrin-coated pits (Miller and Heuser, 1984). However, the fact that in shibire a single gene mutation that probably disrupts receptor-mediated endocytosis causes complete paralysis of neurotransmission argues against multiple, independent endocytotic pathways. Furthermore, endocytosis of synaptic vesicles may require Ca2+, since it is inhibited

Neuron 674

in Ca2+-free media in which neurotransmitter release is stimulated by a-latrotoxin (Ceccarelli and Hurlbut, 1980). Most likely, endocytosis of synaptic vesicles will utilize the same machinery as that used for other pathways involving clathrin-coated pits (reviewed in Pearse and Robinson, 1990). The residence time of synaptic vesicle proteins on the presynaptic plasma membrane after exocytosis seems to be very short, suggesting rapid endocytosis that effectively keeps synaptic vesicle proteins segregated from plasma membrane components. This is in contrast to receptor-mediated endocytosis of receptors in nonneuronal cells, which freely diffuse across the plasma membrane and are trapped in coated pits by an unknown mechanism. Several different brain-specific proteins of coated vesicles have been described (Brodskyand Parham, 1983; Ahle and Ungewickell, 1986,199O; Kohtz and Puszkin, 1988). These may very well have functions that are specifically related to synaptic vesicle endocytosis. Synapses are found only in neurons. Nevertheless, it seems likely that the synaptic vesicle pathway was not invented by the neuron de novo, but that it represents a specialization of a preexisting eukaryotic pathway. Particularly, endocytosis and recycling of synaptic vesicles, but also other aspects of the synaptic vesicle pathway, strongly resemble receptor-mediated endocytosis. Both of these pathways utilize clathrin-coated pits for internalization and recycle as acidic organelles via an endosomal intermediate without contact with the Golgi complex. If the synaptic vesicle pathway is indeed an evolutionary specialization related to receptor-mediated endocytosis, we may expect that homologs of the synaptic vesiclespecific proteins involved in maintainingthis pathway will be found in the nonregulated pathway. Proteins of Synaptic an Organelle

Vesicles:

Architecture

of

Synaptic vesicles are highly specialized and abundant organelles in brain that contain less than 50 major protein components (Jahn and Sudhof, unpublished data). Of these, synaptophysin and the synapsins alone account for approximately 15% of the total vesicle protein, and the synaptobrevins and synaptotagmin seem to be almost as abundant. The most abundant synaptic vesicle proteins that have been molecularly characterized so far (schematically diagrammed in Figure 2) already account for a sizable portion of the total synaptic vesicle protein, suggesting that a description of the majority of the proteins of synaptic vesicles may not be far off. Synaptic vesicles may become the first organelle whose major proteins will be completely characterized, allowing systematic insights into the architecture of a eukaryotic organelle in addition to their implications for neurotransmitter release. For most of the abundant synaptic vesicle proteins, a functional hypothesis was presented. The only ex-

ceptions to this are the synaptobrevins (also referred to as VAMPS [Trimble et al., 19881). These were discovered in Richard Scheller’s laboratory as major components of cholinergic synaptic vesicles from marine rays (Trimble et al., 1988). Independently, Baumert et al. (1989) described the synaptobrevins as rat brain synapticvesicle proteins whose copy number appears to be comparable to that of the synaptophysins (Thiel and Sudhof, unpublished data). The highly conserved four domain structureof the synaptobrevins suggests a role in binding an unidentified protein at the interface between the cytosol and the synaptic vesicle membrane(Siidhof etal., 1989a). However, no binding protein for synaptobrevins has yet been identified. Several general conclusions emerge from the characterization of synaptic vesicle proteins so far. One surprising observation is that the sequences of most of the abundant synaptic vesicle proteins (synapsins, synaptophysins, and synaptobrevins) represent new sequences that are not homologous to currently known proteins. The exceptions to this rule are synaptotagmins, which were the first proteins found to be homologous to the regulatory region of protein kinase C, and rab3A, which was first characterized as a low molecular weight GTP-binding protein of unknown localization. A second generalization is that most of the synaptic vesicle proteins are present in several isoforms. This has been .demonstrated for the synapsins, synaptophysin, synaptobrevin, synaptotagmin, and rab3 (Sudhof et al., 1989c; Elferink et al., 1989;Archer et al., 1990; Knaus et al., 1990; Matsui et al., 1988; Geppert and Sudhof, unpublished data). The high degree of sequence identity between the different isoforms of a synaptic vesicle protein suggests that they are functionally very similar. Most of the isoforms are differentially distributed among synapses, suggesting that their differential expression may confer specialized properties to different synapses. The presence of different isoforms for most synaptic vesicle proteins will make the apparent complexity of the protein pattern of synaptic vesicles higher than it actually is, suggesting that the true number of synaptic vesicle proteins is actually fewer than 50. Furthermore, some synaptic vesicle proteins appear to be highly conserved, as judged by their presence in Drosophila (e.g., synaptotagmin [Perin et al., 1991b, synaptobrevin [Sudhof et al., 1989a], and rab3 [Johnston et al., 1991]), whereas others seem to be poorly conserved in evolution ([e.g., the synapsins and synaptophysin [Sudhof, unpublished data]). How this relates to differences in the functional properties between vertebrate and invertebrate synapses remains to be established. The different synaptic vesicle proteins are attached to the synapticvesicle membrane byavarietyof mechanisms (Figure 2). Different types of membrane proteins are observed (synaptophysin, synaptobrevin, and synaptotagmin) as well as proteins that are attached peripherally to the membrane (the synap-

Review: Synaptic Vesicle Proteins 675

sins) or that are bound to it by a posttranslational modification (rab3A/3B). Some of these proteins presumably become associated with membranes cotranslationallyand others posttranslationally. This finding, together with the fact that no obvious sequence motif, shared by all synaptic vesicle proteins is observed, suggests that the targeting of synapticvesicle proteins does not follow a single simple mechanism. Elucidating the biochemical basis for these multiple targeting mechanisms is the next challenging step, for which the molecular tools are now available.

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