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1.  Cyclical Regulation of the Exocyst and Cell Polarity Determinants for Polarized Cell Growth 
Molecular Biology of the Cell  2005;16(3):1500-1512.
Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.
doi:10.1091/mbc.E04-10-0896
PMCID: PMC551511  PMID: 15647373
2.  The role of Sec3p in secretory vesicle targeting and exocyst complex assembly 
Molecular Biology of the Cell  2014;25(23):3813-3822.
The exocyst has been speculated to mediate the tethering of secretory vesicles to the plasma membrane. However, there has been no direct experimental evidence for this notion. An ectopic targeting strategy is used to provide experimental support for this model and investigate the regulators of exocyst assembly and vesicle targeting.
During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.
doi:10.1091/mbc.E14-04-0907
PMCID: PMC4230786  PMID: 25232005
3.  Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana 
Molecular Biology of the Cell  2013;24(4):510-520.
The exocyst complex localizes to distinct foci at the plasma membrane of Arabidopsis thaliana cells. Their localization at the plasma membrane is insensitive to BFA treatment but is decreased in an exocyst-subunit mutant. In turn, exocyst-subunit mutants show decreased exocytosis.
The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.
doi:10.1091/mbc.E12-06-0492
PMCID: PMC3571873  PMID: 23283982
4.  Rab7A Is Required for Efficient Production of Infectious HIV-1 
PLoS Pathogens  2011;7(11):e1002347.
Retroviruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Rab proteins regulate specific steps in intracellular membrane trafficking by recruiting tethering, docking and fusion factors, as well as the actin- and microtubule-based motor proteins that facilitate vesicle traffic. Using virological tests and RNA interference targeting Rab proteins, we demonstrate that the late endosome-associated Rab7A is required for HIV-1 propagation. Analysis of the late steps of the HIV infection cycle shows that Rab7A regulates Env processing, the incorporation of mature Env glycoproteins into viral particles and HIV-1 infectivity. We also show that siRNA-mediated Rab7A depletion induces a BST2/Tetherin phenotype on HIV-1 release. BST2/Tetherin is a restriction factor that impedes HIV-1 release by tethering mature virus particles to the plasma membrane. Our results suggest that Rab7A contributes to the mechanism by which Vpu counteracts the restriction factor BST2/Tetherin and rescues HIV-1 release. Altogether, our results highlight new roles for a major regulator of the late endocytic pathway, Rab7A, in the late stages of the HIV-1 replication cycle.
Author Summary
Human immunodeficiency virus (HIV) propagation requires the assistance of host cell factors at all stages of the infection cycle. HIV exploits components of the cellular membrane sorting machinery for its assembly, budding and release. Rab GTPases are key regulators of membrane-trafficking events, including exocytosis and endocytosis, in eukaryotic cells. Here we show that the late endosome associated Rab7A plays a major role in HIV-1 replication. We find that Rab7A regulates the production of infectious HIV-1 particles at two critical stages. First, Rab7A is required for efficient Env processing and, thus, for the incorporation of mature HIV-1 envelope glycoproteins into virions. Second, Rab7A contributes to the mechanism that counteracts the restriction imposed on HIV-1 release by the cellular restriction factor BST2/Tetherin that physically tethers viral particles to the plasma membrane of infected cells. Altogether these data highlight new roles for a major player of the late endocytic pathway, Rab7A, in the late stages of the HIV-1 replication cycle.
doi:10.1371/journal.ppat.1002347
PMCID: PMC3207927  PMID: 22072966
5.  A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells 
Molecular Biology of the Cell  2013;24(7):1007-1019.
Rab11a and Rab8 work in conjunction with myosin5B to promote discoidal/fusiform vesicle exocytosis at the apical surface of umbrella cells. It is predicted that similar Rab cascades will be common to other regulated secretory pathways.
Multiple Rabs are associated with secretory granules/vesicles, but how these GTPases are coordinated to promote regulated exocytosis is not well understood. In bladder umbrella cells a subapical pool of discoidal/fusiform-shaped vesicles (DFVs) undergoes Rab11a-dependent regulated exocytosis in response to bladder filling. We show that Rab11a-associated vesicles are enmeshed in an apical cytokeratin meshwork and that Rab11a likely acts upstream of Rab8a to promote exocytosis. Surprisingly, expression of Rabin8, a previously described Rab11a effector and guanine nucleotide exchange factor for Rab8, stimulates stretch-induced exocytosis in a manner that is independent of its catalytic activity. Additional studies demonstrate that the unconventional motor protein myosin5B motor (Myo5B) works in association with the Rab8a–Rab11a module to promote exocytosis, possibly by ensuring transit of DFVs through a subapical, cortical actin cytoskeleton before fusion. Our results indicate that Rab11a, Rab8a, and Myo5B function as part of a network to promote stretch-induced exocytosis, and we predict that similarly organized Rab networks will be common to other regulated secretory pathways.
doi:10.1091/mbc.E12-08-0568
PMCID: PMC3608489  PMID: 23389633
6.  Exocyst Subunits Exo70 and Exo84 Cooperate with Small GTPases to Regulate Behavior and Endocytic Trafficking in C. elegans 
PLoS ONE  2012;7(2):e32077.
The exocyst complex is required for cell polarity regulation and the targeting and tethering of transport vesicles to the plasma membrane. The complex is structurally well conserved, however, the functions of individual subunits and their regulation is poorly understood. Here we characterize the mutant phenotypes for the exocyst complex genes exoc-7 (exo70) and exoc-8 (exo84) in Caenorhabditis elegans. The mutants display pleiotropic behavior defects that resemble those observed in cilia mutants (slow growth, uncoordinated movement, defects in chemo-, mechano- and thermosensation). However, no obvious morphological defects in cilia were observed. A targeted RNAi screen for small GTPases identified eleven genes with enhanced phenotypes when combined with exoc-7, exoc-8 single and exoc-7;exoc-8 double mutants. The screen verified previously identified functional links between the exocyst complex and small GTPases and, in addition, identified several novel potential regulators of exocyst function. The exoc-8 and exoc-7;exoc-8 mutations caused a significant size increase in the rab-10 RNAi-induced endocytic vacuoles in the intestinal epithelial cells. In addition, exoc-8 and exoc-7;exoc-8 mutations resulted in up-regulation of RAB-10 expression and affected the accumulation of endocytic marker proteins in these cells in response to rab-10 RNAi. The findings identify novel, potential regulators for exocyst function and show that exoc-7 and exoc-8 are functionally linked to rab-10 in endosomal trafficking in intestinal epithelial cells in C. elegans.
doi:10.1371/journal.pone.0032077
PMCID: PMC3289633  PMID: 22389680
7.  Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p 
The Journal of Cell Biology  2004;167(5):889-901.
Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.
doi:10.1083/jcb.200408124
PMCID: PMC2172445  PMID: 15583031
8.  The rab Exchange Factor Sec2p Reversibly Associates with the Exocyst 
Molecular Biology of the Cell  2006;17(6):2757-2769.
Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.
doi:10.1091/mbc.E05-10-0917
PMCID: PMC1474791  PMID: 16611746
9.  Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole 
Molecular Biology of the Cell  2012;23(7):1267-1282.
A molecular dissection of contractile vacuole (CV) discharge shows that Rab8a is recruited to the CV a few seconds before the exocyst. Together they tether it to the plasma membrane and commit it to fusion. GTP hydrolysis is necessary for vacuole detethering, a process in which LvsA, a protein of the Chédiak–Higashi family, plays a crucial role.
Water expulsion by the contractile vacuole (CV) in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion, and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a effector, Rab8a, the exocyst complex, and LvsA, a protein of the Chédiak–Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder, suggesting that a Rab8a guanine nucleotide exchange factor activity is associated with the complex. Absence of Drainin leads to overtethering and blocks fusion, whereas expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in postfusion detethering. Of interest, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype.
doi:10.1091/mbc.E11-06-0576
PMCID: PMC3315810  PMID: 22323285
10.  Large-Scale Protein Identification in Intracellular Aquaporin-2 Vesicles from Renal Inner Medullary Collecting Duct 
Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2(AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat IMCD. Immuno-gold EM and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE, followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 (associated with early endosomes), Rab7 (late endosomes), and Rab11 and Rab25 (recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, VAMP2, and VAMP3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network (TGN), components of the exocyst complex, and several motor proteins including myosin IC, non-muscle myosin IIA&B, myosin VI, and myosin IXB. Beyond this, identification of multiple ER-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum (RER). These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, TGN, and RER.
doi:10.1074/mcp.M500049-MCP200
PMCID: PMC1435688  PMID: 15905145
proteomics; endosomes; myosin; vasopressin; mass spectrometry
11.  Phylogeny and evolution of Rab7 and Rab9 proteins 
Background
An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network.
Results
Although Rab7 and Rab9 proteins are quite small and show heterogeneous rates of substitution in different lineages, we found a phylogenetic signal and inferred evolutionary relationships between them. Rab7 proteins evolved before radiation of main eukaryotic supergroups while Rab9 GTPases diverged from Rab7 before split of choanoflagellates and metazoans. Additional duplication of Rab9 and Rab7 proteins resulting in several isoforms occurred in the early evolution of vertebrates and next in teleost fishes and tetrapods. Three Rab7 lineages emerged before divergence of monocots and eudicots and subsequent duplications of Rab7 genes occurred in particular angiosperm clades. Interestingly, several Rab7 copies were identified in some representatives of excavates, ciliates and amoebozoans. The presence of many Rab copies is correlated with significant differences in their expression level. The diversification of analysed Rab subfamilies is also manifested by non-conserved sequences and structural features, many of which are involved in the interaction with regulators and effectors. Individual sites discriminating different subgroups of Rab7 and Rab9 GTPases have been identified.
Conclusion
Phylogenetic reconstructions of Rab7 and Rab9 proteins were performed by a variety of methods. These Rab GTPases show diversification both at the phylogenetic, expression and structural levels. The presence of many Rab7 and Rab9 isoforms suggests their functional specialization and complexity of subcellular trafficking even in unicellular eukaryotes. The identified less conserved regions in analysed Rab sequences may directly contribute to such a differentiation.
doi:10.1186/1471-2148-9-101
PMCID: PMC2693434  PMID: 19442299
12.  The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p 
The Journal of Cell Biology  2006;172(1):55-66.
Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.
doi:10.1083/jcb.200510016
PMCID: PMC2063532  PMID: 16390997
13.  Rab9 GTPase Is Required for Replication of Human Immunodeficiency Virus Type 1, Filoviruses, and Measles Virus 
Journal of Virology  2005;79(18):11742-11751.
Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. By using gene trap insertional mutagenesis, we identified Rab9, which mediates late-endosome-to-trans-Golgi-network trafficking, among several candidate host genes whose disruption allowed the survival of Marburg virus-infected cells, suggesting that Rab9 is utilized in Marburg replication. Although Rab9 has not been implicated in human immunodeficiency virus (HIV) replication, previous reports suggested that the late endosome is an initiation site for HIV assembly and that TIP47-dependent trafficking out of the late endosome to the trans-Golgi network facilitates the sorting of HIV Env into virions budding at the plasma membrane. We examined the role of Rab9 in the life cycles of HIV and several unrelated viruses, using small interfering RNA (siRNA) to silence Rab9 expression before viral infection. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to-trans-Golgi vesicular transport. In addition, silencing studies revealed that HIV replication was dependent on the expression of Rab11A, which mediates trans-Golgi-to-plasma-membrane transport, and that increased HIV Gag was sequestered in a CD63+ endocytic compartment in a cell line stably expressing Rab9 siRNA. Replication of the enveloped Ebola, Marburg, and measles viruses was inhibited with Rab9 siRNA, although the nonenveloped reovirus was insensitive to Rab9 silencing. These results suggest that Rab9 is an important cellular target for inhibiting diverse viruses and help to define a late-endosome-to-plasma-membrane vesicular transport pathway important in viral assembly.
doi:10.1128/JVI.79.18.11742-11751.2005
PMCID: PMC1212642  PMID: 16140752
14.  Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells 
Molecular Biology of the Cell  2007;18(11):4483-4492.
The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P2. Disrupting Exo70-PI(4,5)P2 interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P2-binding–deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.
doi:10.1091/mbc.E07-05-0461
PMCID: PMC2043555  PMID: 17761530
15.  CED-10/Rac1 Regulates Endocytic Recycling through the RAB-5 GAP TBC-2 
PLoS Genetics  2012;8(7):e1002785.
Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.
Author Summary
When cargo is internalized from the cell surface by endocytosis, it enters a series of intracellular organelles called endosomes. Endosomes sort cargo, such that some cargos are sent to the lysosome for degradation, while others are recycled to the plasma membrane. Small GTPase proteins (Rabs) are well-known master regulators of endosome function. As cargo moves through the endosomal system, it must pass from the domain controlled by one Rab-GTPase to the domain controlled by another. Little is known about how transitions along the recycling pathway are controlled, or if Rab transitions are necessary for cargo recycling. Here we identified a group of proteins that act on recycling endosomes to deactivate the early acting GTPase RAB-5. Disruption of any of these proteins interferes with recycling. Our work shows that RAB-5 deactivation is important for cargo recycling, and it provides some of the first mechanistic insight into how changes in Rabs can be controlled during endocytic recycling. Importantly, several proteins that we found contribute to this recycling function have roles in other cellular processes, such as cell migration and the removal of cell corpses. Therefore our work also suggests that endocytic recycling could contribute to these processes in previously unsuspected ways.
doi:10.1371/journal.pgen.1002785
PMCID: PMC3395619  PMID: 22807685
16.  Structural Insights into a Unique Legionella pneumophila Effector LidA Recognizing Both GDP and GTP Bound Rab1 in Their Active State 
PLoS Pathogens  2012;8(3):e1002528.
The intracellular pathogen Legionella pneumophila hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling.
Author Summary
Legionella pneumophila delivers 275 validated substrates into the host cytosol by its Dot/Icm type IV secretion system. Several substrates including SidM/DrrA and LidA directly interact with the host Rab GTPases and interfere with the vesicle secretion pathway. SidM/DrrA is necessary for Rab1 recruitment, function as a Rab1 specific GDI displacement factor and guanine nucleotide exchange factor. LidA has the auxiliary activity for Rab1 recruitment, whereas it is more important for the formation of the replication vacuole compared with SidM/DrrA. LidA is predicted to be the first substrate secreted by the Dot/Icm system and is critical for maintaining the integrity of the bacterial cell. Moreover, it expresses throughout the intracellular growth phase, localizes to early secretory compartments, and interacts with several members of Rab family. Here we present the crystal structures of LidA coiled-coil domain in complex with two different states of Rab1, GDP- and GTP-bound. The GDP-bound Rab1 in the complex surprisingly has the same conformation with the GTP-bound Rab1, revealing that LidA can retain Rab1 persistently in its active state. Our structures add a new insight into the regulation of the host Rab1 membrane cycle by pathogen-secreted coiled-coil effector.
doi:10.1371/journal.ppat.1002528
PMCID: PMC3295573  PMID: 22416225
17.  GTP Hydrolysis of TC10 Promotes Neurite Outgrowth through Exocytic Fusion of Rab11- and L1-Containing Vesicles by Releasing Exocyst Component Exo70 
PLoS ONE  2013;8(11):e79689.
The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis.
doi:10.1371/journal.pone.0079689
PMCID: PMC3817099  PMID: 24223996
18.  From Sorting Endosomes to Exocytosis: Association of Rab4 and Rab11 GTPases with the Fc Receptor, FcRn, during RecyclingV⃞ 
Molecular Biology of the Cell  2005;16(4):2028-2038.
A longstanding question in cell biology is how is the routing of intracellular organelles within cells regulated? Although data support the involvement of Rab4 and Rab11 GTPases in the recycling pathway, the function of Rab11 in particular is uncertain. Here we have analyzed the association of these two Rab GTPases with the Fc receptor, FcRn, during intracellular trafficking. This Fc receptor is both functionally and structurally distinct from the classical Fcγ receptors and transports immunoglobulin G (IgG) within cells. FcRn is therefore a recycling receptor that sorts bound IgG from unbound IgG in sorting endosomes. In the current study we have used dual color total internal reflection fluorescence microscopy (TIRFM) and wide-field imaging of live cells to analyze the events in human endothelial cells that are involved in the trafficking of FcRn positive (FcRn+) recycling compartments from sorting endosomes to exocytic sites at the plasma membrane. Our data are consistent with the following model for this pathway: FcRn leaves sorting endosomes in Rab4+Rab11+ or Rab11+ compartments. For Rab4+Rab11+ compartments, Rab4 depletion occurs by segregation of the two Rab proteins into discrete domains that can separate. The Rab11+FcRn+ vesicle or tubule subsequently fuses with the plasma membrane in an exocytic event. In contrast to Rab11, Rab4 is not involved in exocytosis.
doi:10.1091/mbc.E04-08-0735
PMCID: PMC1073680  PMID: 15689494
19.  TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans 
PLoS Genetics  2012;8(5):e1002722.
Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.
Author Summary
Synaptic transmission is mainly mediated by the triggered release of neurotransmitters from synaptic vesicles (SVs). To regulate synaptic transmission and neuronal activity, neurons also release neuropeptides and hormones from dense core vesicles (DCVs). While SVs can be recycled locally at the synapse, DCVs have to be newly synthesized in the cell body after release. The formation of new DCVs requires a multi-step maturation process. During this maturation, the neuropeptides are processed into their active form and factors that would disturb DCV release are removed. Only properly matured DCVs are able to undergo efficient release after stimulation. Since DCV biogenesis mainly uses the normal secretory pathway, an elaborate machinery must exist that guarantees efficient sorting and retention of DCV cargo. Previously, we identified the small GTPase RAB-2 and its effector RIC-19/ICA69 to be involved in the retention of soluble cargo in DCVs. In a screen for molecules that regulate RAB-2 activity during DCV maturation, we identified the evolutionarily conserved TBC domain-containing protein, TBC-8. We demonstrate that TBC-8 is a putative RAB-2 GAP, which also binds to RIC-19/ICA69. Thus, RAB-2 might recruit its own GAP via its effector RIC-19, which suggests that a highly dynamic cycling of RAB-2 is required for DCV maturation.
doi:10.1371/journal.pgen.1002722
PMCID: PMC3359978  PMID: 22654674
20.  Trans-Golgi network localized small GTPase RabA1d is involved in cell plate formation and oscillatory root hair growth 
BMC Plant Biology  2014;14(1):252.
Background
Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described.
Results
The transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal compartments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking.
Conclusions
RabA1d is located in early endosomes/TGN and is involved in vesicle trafficking. RabA1d participates in both cell plate formation and root hair oscillatory tip growth. The specific GFP-RabA1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0252-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0252-0
PMCID: PMC4180857  PMID: 25260869
Arabidopsis; Cell plate; Cytokinesis; Rab GTPase; Localization; RabA1d; Root hair; Trans-Golgi network; Vesicle
21.  Functional Cross-Talk between Rab14 and Rab4 through a Dual Effector, RUFY1/Rabip4 
Molecular Biology of the Cell  2010;21(15):2746-2755.
Rab14 binds in a GTP-dependent manner to RUFY1/Rabip4, which had been originally identified as a Rab4 effector. We suggest that Rab14 and Rab4 act sequentially; Rab14 is required for recruitment of RUFY1 onto endosomes and subsequent RUFY1 interaction with Rab4 may allow endosomal tethering and fusion.
The small GTPase Rab14 localizes to early endosomes and the trans-Golgi network, but its cellular functions on endosomes and its functional relationship with other endosomal Rab proteins are poorly understood. Here, we report that Rab14 binds in a GTP-dependent manner to RUFY1/Rabip4, which had been originally identified as a Rab4 effector. Rab14 colocalizes well with Rab4 on peripheral endosomes. Depletion of Rab14, but not Rab4, causes dissociation of RUFY1 from endosomal membranes. Coexpression of RUFY1 with either Rab14 or Rab4 induces clustering and enlargement of endosomes, whereas a RUFY1 mutant lacking the Rab4-binding region does not induce a significant morphological change in the endosomal structures even when coexpressed with Rab14 or Rab4. These findings suggest that Rab14 and Rab4 act sequentially, together with RUFY1; Rab14 is required for recruitment of RUFY1 onto endosomal membranes, and subsequent RUFY1 interaction with Rab4 may allow endosomal tethering and fusion. Depletion of Rab14 or RUFY1, as well as Rab4, inhibits efficient recycling of endocytosed transferrin, suggesting that Rab14 and Rab4 regulate endosomal functions through cooperative interactions with their dual effector, RUFY1.
doi:10.1091/mbc.E10-01-0074
PMCID: PMC2912359  PMID: 20534812
22.  The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes 
Cellular microbiology  2010;12(9):1292-1307.
Summary
Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV’s selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A, and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase-deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A, and GFP-Rab11A localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.
doi:10.1111/j.1462-5822.2010.01468.x
PMCID: PMC2923681  PMID: 20345488
23.  RIM, Munc13, and Rab3A Interplay in Acrosomal Exocytosis 
Experimental Cell Research  2012;318(5):478-488.
Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have a central role in membrane docking.
doi:10.1016/j.yexcr.2012.01.002
PMCID: PMC3288645  PMID: 22248876
RIM; Munc13; Rab3A; acrosome reaction; membrane docking; human sperm
24.  Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack 
The Journal of Cell Biology  1994;126(6):1393-1406.
The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway.
PMCID: PMC2290957  PMID: 8089173
25.  Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes 
The Journal of Cell Biology  2012;198(4):545-560.
Rab10 activation promotes GLUT4 storage vesicle recruitment to the plasma membrane in response to insulin and coordinates with myosin-Va to mediate vesicle fusion.
Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.
doi:10.1083/jcb.201111091
PMCID: PMC3514028  PMID: 22908308

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