Quantitative Analysis of Synaptic Vesicle Rabs Uncovers Distinct Yet Overlapping Roles for Rab3a and Rab27b in Ca²⁺-Triggered Exocytosis

Abstract

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca 2ϩ -triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.

Distinct yet overlapping roles of Rab GTPases on synaptic vesicles

“…We have recently resolved the identity and number of Rab proteins enriched on highly purified SVs. 6 Here we revisit these findings alongside past is by far the most abundant variant at the synapse, accounting for some 25% of total brain Rab expression and dominating up to 2.5% of the SV surface (equating to ~10 copies/vesicle). 3 Therefore, it was not unexpected that Rab3a headed our list of Rabs highly enriched on purified SVs.…”

“…6 Combining chemical labelling (iTRAQ TM ) with state-of-the-art proteomics, we were able to distinguish between those Rabs that were specifically enriched on SVs and those which were not. This approach enabled us to quantitatively monitor individual Rab enrichment profiles over multiple membrane and cytosolic fractions obtained at specific stages of the SV purification procedure (thereby circumventing problems that are inherent in proteomic data sets from single fractions).…”

“…Indeed, we demonstrated that Rab27b, but not Rab27a, was also abundantly enriched on SVs by immuno-analyses. 6 This exquisite expression pattern followed on SVs encompassing those whose roles are well established in exocytic (Rab3a/ b/c and Rab27b) and endocytic (Rab4b, Rab5a, Rab10, Rab11b and Rab14) transport. Intriguingly, many other Rabs were also found to be moderately enriched on purified SVs although not to the same extent as those discussed above.…”

Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis

“…These "secretory Rabs" localize to vesicles and secretory granules in a variety of secretory cell types (1,8,(13)(14)(15). They control the recruitment, tethering, and/or docking of secretory vesicles to the plasma membrane through interaction with effectors (16).…”

“…In the most recent study analyzing Rab3 and Rab27 simultaneously, both proteomic and quantitative Western blot analyses of subcellular fractions prepared from rat brain homogenates demonstrated the marked enrichment of three isoforms of Rab3 (A, B, and C), as well as Rab27B, on synaptic vesicles. Subpopulations of Rab3A molecules also localize to brain cytosol and plasma membrane, whereas Rab27B is not found in the cytosol (15). In a recent review, the authors hypothesized that the roles of Rab3A and Rab27B in the synaptic vesicle cycle are complementary, and that these isoforms act in successive steps (22).…”