Better Late Than Never: A Role for Rabs Late in Exocytosis
Authors: Andrew J. Bean, Richard H. Scheller
Journal: Neuron (1997)

Since the discovery of the rab family of small GTP-binding proteins in yeast screens for vesicle trafficking mutants, a search has been underway for a precise definition of their role in the secretory pathway. The rab proteins comprise a homologous, large (∼40 mammalian proteins, 11 in yeast) branch of the ras superfamily whose members are present at all trafficking steps throughout the secretory pathway (8Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (655) Google Scholar). Some rab proteins are differentially expressed and may therefore have specific roles in certain cell types. Rab3a and its isoform, rab3c, are expressed exclusively in neurons and neuroendocrine cells and have therefore garnered considerable interest because of their possible role in regulated secretion. In the past few years, our insight into the molecular mechanisms of exocytosis has increased, although it has remained difficult to place rab3a in the context of other proteins known to be essential for regulated secretion. Thus far it has only been possible to assign rabs a mechanistically undefined role in the docking/fusion of vesicles with their target membranes. This review focuses on three interesting and important papers (1Castillo P.E. Janz R. Sudhof T.C. Tzounopoulos T. Malenka R.C. Nicoll R.A. Nature. 1997; 388: 590-593Crossref PubMed Scopus (288) Google Scholar, 3Geppert M. Goda Y. Stevens C.F. Sudhof T.C. Nature. 1997; 387: 810-814Crossref PubMed Scopus (335) Google Scholar, 10Wang Y. Okamoto M. Schmitz F. Hofmann K. Sudhof T.C. Nature. 1997; 388: 593-598Crossref PubMed Scopus (511) Google Scholar) that suggest that rab3a may be involved in the final stages of the exocytic event and that a new rab3a effector protein may provide a clue to the rab3a mechanism. The process of vesicular neurotransmitter release involves targeted trafficking of the vesicles to the presynaptic release site or active zone, docking/priming of vesicles to be released, fusion of the vesicle and plasma membrane bilayers, and recycling of the vesicular membrane (4Hay J.C. Scheller R.H. Curr. Opin. Cell Biol. 1997; 9: 505-512Crossref PubMed Scopus (248) Google Scholar). Initial attempts to understand the molecular basis of vesicular release involved the characterization of proteins present on the synaptic vesicles and on the presynaptic plasma membrane. Subsequent studies revealed that protein complexes that form between integral components of the synaptic vesicle (v-SNAREs) and the plasma membrane (t-SNAREs) serve as receptors for soluble and peripheral membrane proteins, apparently acting as general secretory factors in multiple transport steps. Related sets of SNARE proteins, presumably all undertaking similar functions, are present at each step of the secretory pathway and have been suggested to encode the specificity of vesicle transport. Unique rab proteins are also associated with each step in the secretory pathway, suggesting that they too may perform a common function and yet confer specificity to each trafficking step. Genetic analysis of the yeast constitutive secretory pathway has converged with progress on synaptic proteins, and mammalian homologs of most of the essential yeast secretory proteins, including rabs, have been identified (4Hay J.C. Scheller R.H. Curr. Opin. Cell Biol. 1997; 9: 505-512Crossref PubMed Scopus (248) Google Scholar). Biochemical studies and in vitro reconstitution of transport steps have indicated a requirement for the rab proteins but have yet to provide a clear common function. The function of the rab proteins is likely dependent on their enzyme activity as well as their effectors. Much has been learned about the possible function of the rab GTPase activity and the proteins involved in the rab GDP-GTP cycle from studies on many different rab proteins, with the assumption that this activity and its function are common to all rabs (9Sudhof T.C. Neuron. 1997; 18: 519-522Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (655) Google Scholar). The cycling of rab proteins from the GDP-bound state to the GTP-bound state appears to correspond with association of the rab proteins with their target organelles. The search for rab effectors has proven somewhat more elusive, and although many new proteins have recently been discovered as rab-binding proteins using the two-hybrid approach, they lack a common structure that would allow coherent speculation about a common function. Rabphilin 3a was the first identified rab effector (8Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (655) Google Scholar). Rabphilin 3a is a GTP-dependent rab3a-binding protein associated with the synaptic vesicle that possesses a zinc finger and two C2 domains. Rabphilin 3a also interacts with a-actinin, an interaction that has led to the speculation that it may play a role in rab3a-dependent cytoskeletal remodeling. If all rab proteins have a similar function in vesicle trafficking, and genetic analysis of the yeast rab genes SEC4 and YPT1 has pointed out genetic interactions with known components of the SNARE machinery, why has it been so difficult to observe interactions of rabs with the vesicle trafficking machinery? It is likely that these interactions have only been rarely observed either because rabs do not directly bind to these proteins, or because rab interactions are very transient. In the few instances where rab–SNARE interactions have been observed, rabs appear to enable SNARE complex assembly (5Lian J.P. Stone S. Jiang Y. Lyons P. Ferro-Novick S. Nature. 1994; 372: 698-701Crossref PubMed Scopus (159) Google Scholar, 6Lupashin V.V. Waters M.G. Science. 1997; 276: 1255-1258Crossref PubMed Scopus (182) Google Scholar). While direct binding of YPT1 to the t-SNARE Sed5 has been reported, this interaction is not observed at other trafficking steps and remains controversial. In the absence of the ability to place rabs in a biochemical pathway that includes known vesicle trafficking machinery, one approach is simply to remove one of the rab proteins from cells and examine the resultant phenotype. In 1994, Geppert et al. reported on the removal of the rab3a gene from mice by homologous recombination (2Geppert M. Bolshakov V.Y. Siegelbaum S.A. Takei K. De Camilli P. Hammer R.E. Sudhof T.C. Nature. 1994; 369: 493-497Crossref PubMed Scopus (395) Google Scholar). The homozygous mutant mice express no detectable rab3a protein, and levels of other rab3 proteins as well as 18 other synaptic proteins are not altered. The only protein level observed to change in the rab3a mutants is that of the rab3a effector protein, rabphilin 3a (70% decrease). An initial physiological study of these rab3a mutant mice suggested that rab3a is not essential for synaptic vesicle exocytosis (2Geppert M. Bolshakov V.Y. Siegelbaum S.A. Takei K. De Camilli P. Hammer R.E. Sudhof T.C. Nature. 1994; 369: 493-497Crossref PubMed Scopus (395) Google Scholar). Different physiological parameters were examined in hippocampal slices taken from homozygous mutants and wild-type isogenic mice. Recording from CA1 pyramidal cells demonstrated that there were no detectable differences in excitatory postsynaptic currents (EPSCs) upon stimulation of Schaffer collateral/commissural fibers and no alterations in plasticity-related measures such as paired-pulse facilitation, post-tetanic potentiation, or long-term potentiation (LTP). It was only after short trains of repetitive stimulation that a difference between wild type and rab3a mutants could be observed. Following 10–15 stimuli, the size of the resultant EPSCs was depressed in the mutants compared to the wild-type mice. These studies suggested that the rab3a mutant phenotype is appreciated only after the predocked vesicle supply is exhausted (although the lack of effect on LTP is not clear; see below), and the authors favored the hypothesis that rab3a functions either in recruiting vesicles from a reserve pool and/or in subsequent vesicle docking. This result is consistent with the phenotype of the yeast SEC4 mutant, which produces a buildup of post-Golgi vesicles that are apparently unable to dock with the plasma membrane. One of the recent papers reexamined the phenotype of the rab3a mutant mouse (3Geppert M. Goda Y. Stevens C.F. Sudhof T.C. Nature. 1997; 387: 810-814Crossref PubMed Scopus (335) Google Scholar). Using cultured hippocampal neurons from mutant and wild-type mice, these authors found no difference in the amplitude or spontaneous rate of individual quantal events. Moreover, estimation of the size of the readily releasable pool of vesicles and the vesicle pool refilling rate revealed no differences between wild-type and mutant cells. Since the quantal properties of transmitter release and the vesicle pool size and refilling rate are all unaffected by the absence of rab3a, these authors posited that rab3a must be involved in a post-docking event. By examining the quantal content evoked by single stimuli, it was observed that the evoked quantal release is increased 100% in rab3a mutant cells compared to wild type. Thus, in the absence of rab3a, a single stimulus results in an increase in vesicle fusion events per synapse. This result and its interpretation suggested that paired-pulse facilitation should be enhanced in rab3a mutant mice, and that is what these authors observed. The increase in evoked release observed in rab3a mutants could either be due to an increase in the probablilty of releasing one quantum per stimulus or to an increase in the number of quanta released when a release event occurs. To try to differentiate between these possibilities, the authors examined the kinetics of inhibition of synaptic responses in wild-type and mutant mice produced by MK-801, a long lasting open-channel blocker of NMDA-type glutamate receptors. Thus, as more quanta are released, they will open postsynaptic glutamate receptor channels, allowing MK-801 to progressively block synaptic currents. The result was that the kinetics of MK-801 blockade were not different between wild type and rab3a mutants. However, since the authors already knew that evoked quantal release is increased in rab3a mutants, they reasoned that if increased vesicle fusion occurs at synapses that already release one quantum (enough to saturate postsynaptic NMDA receptors), then they might not observe a change in MK-801 blockade kinetics. The final experiment of this report was to examine the MK-801 blockade kinetics again, but this time in the presence of a small concentration of the competitive NMDA antagonist, APV. The addition of APV reduced the apparent glutamate concentration, thus slowing the MK-801 blocking rate and therefore increasing the resolution of their assay. Under these conditions, the MK-801 blocking rate was found to be faster in rab3a mutant cells than in wild-type cells. These data suggest that the overall probablilty of release at all synapses is not increased in rab3a mutants but that the increase observed preferentially occurs at synapses that are already releasing a quantum. These results led the authors to speculate that the function of rab3a is to limit release to a single quantum per release site by inhibiting mutivesicle fusion (Figure 1). A mechanism was hypothesized in which a single vesicle fusion event triggers a destabilization of the active zone by insertion of the membrane from the fused vesicle. The role of rab3a on fusion could be either positive or negative; rab3a could facilitate fusion and in that way promote destabilization of the active zone, or rab3a could inhibit fusion through regulated interactions with an effector such as RIM (see below) that could alter the active zone architecture. The exact mechanism by which this destabilization would occur is unclear. A mechanical disruption producing a ripple or wave in the plasma membrane, resulting in an inability of fusion proteins to act, might yield a localized area of inhibition surrounding a fusion event, but this is certainly an unprecedented mechanism. If the above model turns out to be correct, it would appear that the function of rab3a is synapse specific, as other trafficking steps are unlikely to have this property. Does this mean that each of the 40 rab proteins has a unique function? This idea is unsettling in an age when so much of biology is converging on a set of common biochemical mechanisms. Perhaps a common rab mechanism produces distinct properties at various trafficking steps based on unique characteristics of the different stages of the secretory pathway. In a continuing search for rab3a effectors, Wang et al. have used the yeast two-hybrid approach to discover a novel rab3a-binding protein called RIM (Rab Interacting Molecule10Wang Y. Okamoto M. Schmitz F. Hofmann K. Sudhof T.C. Nature. 1997; 388: 593-598Crossref PubMed Scopus (511) Google Scholar). The RIM protein binds to rab3a in a GTP-dependent manner and not to any other rab proteins examined. RIM possesses a zinc finger and a C2 domain as well as a PDZ domain. There are many C2 domain-containing proteins in the nerve terminal (e.g., synaptotagmin, doc-2), suggesting that this is a widely used protein–lipid/protein–protein interaction motif in the synapse. The zinc finger in RIM is similar in structure to that expressed in other proteins involved in protein transport, including the rab 3a effector rabphilin as well as the SNAP-25 interacting proteins Hrs-2, EEA-1, and Vac1. Interestingly, both RIM and rabphilin bind to rab3a through sequences contained within the zinc finger domain, suggesting that other proteins with this domain may also interact with rab proteins. The localization of RIM reveals perhaps its most intriguing feature: RIM appears to be localized to the presynaptic terminal, and at least some of the RIM protein is associated with the active zone. The localization of RIM to the ribbons in photoreceptor terminals further suggests a specialized localization at the site of vesicle docking and fusion. In PC12 cells, an N-terminally truncated RIM protein expressing only the rab3a-binding domain enhanced the calcium-dependent secretion of cotransfected growth hormone. In the same assay, Rab3a inhibited secretion of growth hormone, and both rab3a and N-terminal RIM protein expressed together were not different from control. These data suggest that RIM, possibly by interfering with rab3a function, may promote transmitter release. However, as with many of the newly discovered synaptic proteins, thus far no functional studies with RIM have been performed in neurons. The discovery of RIM provides rab3a with another effector that is localized to a cellular compartment distinct from rabphilin 3a, suggesting that multiple rab3a effectors could operate in series, triggering multiple downstream signals. Whereas rabphilin 3a interacts with GTP-rab3a on the vesicle, RIM appears ideally situated to be involved in later steps in the transport pathway (Figure 2). Thus, the rab3a-RIM complex, perhaps regulated by GTP hydrolysis or RIM phosphorylation, could play a role in rab3a-induced inhibition of multivesicular release. Further experiments are underway to test this hypothesis, and through the merger of genetics, biochemistry, and physiology, we will understand more about the mysteries of the transmitter release machinery. Do these studies suggest that rab3a has a small modulatory role on transmitter release that can only be observed using sophisticated approaches that enhance small alterations in the release process? Perhaps not, but you may have to know where to look. The initial physiological study of the rab3a mutant mice reported no alteration in CA1 LTP compared to wild-type mice. Now a study (1Castillo P.E. Janz R. Sudhof T.C. Tzounopoulos T. Malenka R.C. Nicoll R.A. Nature. 1997; 388: 590-593Crossref PubMed Scopus (288) Google Scholar) has examined mossy fiber LTP and found that this form of LTP is absent in rab3a mutant mice. The major difference between these studies is that the former study (no effect) examined LTP in the CA1 area of the hippocampus, where the locus of LTP expression probably has both pre- and postsynaptic components, while the latter study (LTP absent) investigated LTP in the mossy fiber synapse, where LTP is likely expressed presynaptically. Other aspects of mossy fiber presynaptic function, such as the extent of the enhancement of synaptic transmission immediately following the tetanus used to produce LTP, were not altered in rab3a mutant mice. In addition1Castillo P.E. Janz R. Sudhof T.C. Tzounopoulos T. Malenka R.C. Nicoll R.A. Nature. 1997; 388: 590-593Crossref PubMed Scopus (288) Google Scholar report that in mossy fibers, when 20 or 40 ms interpulse intervals are used, there is no increase in paired-pulse facilitation in rab3a mutant mice. This observation is similar to that of 2Geppert M. Bolshakov V.Y. Siegelbaum S.A. Takei K. De Camilli P. Hammer R.E. Sudhof T.C. Nature. 1994; 369: 493-497Crossref PubMed Scopus (395) Google Scholar, who reported no facilitation in CA1 at stimulus intervals >50 ms. However, in a more recent paper3Geppert M. Goda Y. Stevens C.F. Sudhof T.C. Nature. 1997; 387: 810-814Crossref PubMed Scopus (335) Google Scholar have observed facilitation in the rab3a mutants with intervals ranging from 15 to 100 ms. Resolution of these relatively minor inconsistancies awaits further experimentation. Thus, there appears to be a remarkably selective deficit on presynaptic function in rab3a mutant mice, an effect on LTP in the absence of alterations to short term plasticity (paired-pulse, frequency, and post-tetanic facilitation) that would normally be consistent with changes in transmitter release probability. 1Castillo P.E. Janz R. Sudhof T.C. Tzounopoulos T. Malenka R.C. Nicoll R.A. Nature. 1997; 388: 590-593Crossref PubMed Scopus (288) Google Scholar propose a model suggesting that in the absence of rab3a, protein kinase A (thought to be a necessary component in the signaling pathway used to express LTP) would be unable to act, perhaps because its relevant substrates for LTP are rab3a effectors. However, when Sp-8-Br-CPT-cAMPS is used to elevate cAMP levels, the increases in synaptic transmission observed in wild-type and rab3a mutant mice are nearly identical. It is unclear why, under these conditions, increases in synaptic transmission that are proposed to be dependent on the elevation of cAMP working through rab3a are normal in rab3a mutant mice. This inconsistency challenges the hypothesis, and further investigations are clearly required to sort out the mechanism of the increase in neurotransmission produced during mossy fiber LTP. The generation of rab3a mutant mice has led to three physiological studies concerned with aspects of presynaptic function, producing three results that are consistent with regard to a deficit in presynaptic release. However, the extent and nature of the deficits observed are dependent on the synapses investigated and the protocols used for the studies. Together, these studies make important progress in understanding mechanisms of synaptic plasticity and rab3a action, while at the same time raising some broad questions regarding rab function. Most generally, as discussed earlier, do all rabs have a similar function as is currently thought for SNAREs? Regarding the mechanism of rab function, invoking a process dependent on a rab-binding protein is appealing; however, there are no yeast rabphilin or RIM homologs, and yeast certainly use rabs for vesicle trafficking. Perhaps each vesicle trafficking step will turn out to have a unique set of regulators, and the function of the rab proteins is to control, in time and space, the association of a heterogenous set of effectors. Thus, these recent studies have suggested new ways to think about the possible function of rabs, but the final word in the rab function debate has not yet been released.‡To whom correspondence should be addressed.