br Conclusions and perspectives br Acknowledgements This wor

Conclusions and perspectives
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 21802173, 21405182 and 21773315), the Natural Science Foundation of Guangdong Province (Grant Nos. 2018A030310301, 201710010019 and 2014A030313232, 201804020025), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2017) and Pearl River S&T Nova Program of Guangzhou (201710010019), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program, Guangdong Provincial Key Platform and Major Scientific Research Projects for Colleges and Universities (2015KCXTD029). Many thanks to Dr. Manon Guille-Collignon and Dr. Frédéric Lemaître for their careful reading and valuable suggestions.
Introduction Rabs are guanine nucleotide-dependent molecular switches that regulate intracellular trafficking and define the identity of subcellular membrane domains. Rabs THZ1 Hydrochloride between inactive, GDP-bound and active, GTP-bound states. Because they cannot efficiently interconvert from one form to the other, the switch mechanism is controlled by accessory proteins: guanine nucleotide exchange factors (GEFs) that mediate their activation by favoring the release of GDP (GTP enters the empty Rab because its cytosolic concentrations are higher than those of GDP [60]) and GTPase-activating proteins that exert their activity by accelerating hydrolysis of GTP. In their active state, Rabs interact with specific effectors [49,58]. Recent proteomics analysis detected a large number of Rab isoforms on purified secretory vesicles; some of them are, in addition, known residents of intracellular structures that are not part of the secretory pathway. Only members of the 3 and 27 subfamilies are classified as secretory since they localize specifically to mature dense-core secretory vesicles and control regulated exocytosis by recruiting and docking of secretory vesicles to the plasma membrane (reviewed in [27]). GRAB (also known as Rab3A interacting protein (rabin3)-like 1 or Rab3IL1) has been described as a guanine nucleotide exchange factor for Rab3A. It exhibits GEF activity toward Rab3A both in vitro and in vivo in PC12 cells, where overexpression of GRAB augments — and transfection with antisense RNA targeting GRAB decreases — the loading of endogenous Rab3A with GTP. GRAB-transfected PC12 and bovine adrenal chromaffin cells exhibit the same secretion phenotype as cells transfected with Rab3A, which is consistent with the premiss that GRAB is a physiologic GEF for Rab3 [44]. GRAB's GEF catalytic activity resides in a coiled-coil sequence (amino acids 70–160) highly homologous to the yeast protein Sec2. This is a GEF for Sec4, a yeast GTPase involved in secretion whose mammalian orthologues are Rabs3 and 8. Rabphilin3a was originally described as a Rab3A effector expressed in neuroendocrine cells [15]. Effectors are defined through their ability to bind to a specific Rab selectively in its GTP-bound state and mediate at least one of its downstream effects. The initial claim that Rabphilin3a is a Rab3 effector was challenged by the discoveries that it is not essential for the regulatory functions of Rab3 in synaptic transmission [55] and that its interaction with Rab3 is weaker than that with Rab27A/B [25,29]. Thus, Rabphilin3a has been subsequently reclassified as a Rab27 effector (for reviews on Rabphilin3a and other Rab27 effectors in somatic cells, see [26,28]). Rab GTPases that work sequentially on a given traffic pathway must be activated at the right time and place to fulfill their functions in an orchestrated fashion. Membrane flow from one organelle to another is coupled to the transition through different Rab-defined compartments. One way in which these proteins accomplish such coordination is through RabGEF cascades, where a GEF is recruited by the first Rab to activate the second Rab acting further down the pathway. This mechanism has been described in yeast (for instance the cascade Ypt31/32 → Sec2 → Sec4, where Sec2 is a Sec4GEF) and mammals (Rab11 → Rabin8 → Rab8, where Rabin8 is a Rab8GEF) [1,31,37,39,50].