The Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for dendrite formation in melanocytes

Vacuolar protein sorting 9 (VPS9)-ankyrin-repeat protein (Varp) has recently been identified as an effector molecule for two small GTPases-Rab32 and Rab38-in the transport of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes in melanocytes. Although Varp contains a Rab21-guanine nucleotide exchange factor (GEF) domain (i.e., VPS9 domain), since Rab21-GEF activity is not required for Tyrp1 transport, nothing is known about the physiological significance of the Rab21-GEF activity in melanocytes. Here we show by knockdown-rescue experiments that the Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for forskolin-induced dendrite formation of cultured melanocytes. We found that Varp-deficient cells are unable to extend dendrites in response to forskolin stimulation and that reexpression of wild-type Varp or a Rab32/38-binding-deficient mutant Varp(Q509A/Y550A) in Varp-deficient cells completely restores their ability to form dendrites. By contrast, VPS9 mutants (D310A and Y350A) and a vesicle-associated membrane protein 7 (VAMP7)-binding-deficient mutant were unable to support forskolin-induced dendrite formation in Varp-deficient cells. These findings indicate that the Rab21-GEF activity and Rab32/38 binding activity of Varp are required for different melanocyte functions, that is, Rab21 activation by the VPS9 domain is required for dendrite formation, and the Rab32/38 effector function of the ankyrin repeat 1 domain is required for Tyrp1 transport to melanosomes, although VAMP7-binding ability is required for both functions.     

Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.     

Multiple Roles of VARP in Endosomal Trafficking: Rabs,Retromer Components and R‐SNARE VAMP7 Meet on VARP

VARP (VPS9‐ankyrin‐repeat protein, also known as ANKRD27) was originally identified as an N‐terminal VPS9 (vacuolar protein sorting 9)‐domain‐containing protein that possesses guanine nucleotide exchange factor (GEF) activity toward small GTPase Rab21 and contains two ankyrin repeat (ANKR) domains in its central region. A number of VARP‐interacting molecules have been identified during the past five years, and considerable attention is now being directed to the multiple roles of VARP in endosomal trafficking. More specifically, VARP is now known to interact with three different types of key membrane trafficking regulators, i.e. small GTPase Rabs (Rab32, Rab38 and Rab40C), the retromer complex (a sorting nexin dimer, VPS26, VPS29 and VPS35) and R‐SNARE VAMP7. By binding to several of these molecules, VARP regulates endosomal trafficking, which underlies a variety of cellular events, including melanogenic enzyme trafficking to melanosomes, dendrite outgrowth of melanocytes, neurite outgrowth and retromer‐mediated endosome‐to‐plasma membrane sorting of transmembrane proteins.      

RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins

Rab GTPases regulate all steps of membrane trafficking. Their interconversion between active, GTP-bound states and inactive, GDP-bound states is regulated by guanine nucleotide exchange factors and GTPase-activating proteins. The substrates for most Rab GTPase-activating proteins (GAPs) are unknown. Rab9A and its effectors regulate transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. We show here that RUTBC1 is a Tre2/Bub2/Cdc16 domain-containing protein that binds to Rab9A-GTP both in vitro and in cultured cells, but is not a GTPase-activating protein for Rab9A. Biochemical screening of RUTBC1 Rab protein substrates revealed highest in vitro GTP hydrolysis-activating activity with Rab32 and Rab33B. Catalysis required Arg-803 of RUTBC1, and RUTBC1 could activate a catalytically inhibited Rab33B mutant (Q92A), in support of a dual finger mechanism for RUTBC1 action. Rab9A binding did not influence GAP activity of bead-bound RUTBC1 protein. In cells and cell extracts, RUTBC1 influenced the ability of Rab32 to bind its effector protein, Varp, consistent with a physiological role for RUTBC1 in regulating Rab32. In contrast, binding of Rab33B to its effector protein, Atg16L1, was not influenced by RUTBC1 in cells or extracts. The identification of a protein that binds Rab9A and inactivates Rab32 supports a model in which Rab9A and Rab32 act in adjacent pathways at the boundary between late endosomes and the biogenesis of lysosome-related organelles.     

Role of Varp,a Rab21 exchange factor and TI‐VAMP/VAMP7 partner,in neurite growth

The vesicular soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin‐insensitive vesicle‐associated membrane protein (TI‐VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI‐VAMP interacts with the Vps9 domain and ankyrin‐repeat‐containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI‐VAMP and Rab21 co‐localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI‐VAMP.     

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.     

Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes

A mutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype "chocolate" (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38(G19V) is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.     

Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.     

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome‐related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC‐1, ‐2, ‐3, or AP‐1, ‐3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A‐depletion phenotype resembles Rab38/32‐inactivated or BLOC‐3‐deficient melanocytes, suggesting that Rab9A works in line with BLOC‐3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC‐3‐deficiency in melanocytes decreased the length of STX13‐positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co‐regulatory GTPases control STX13‐mediated cargo delivery to maturing melanosomes.     

Identification and characterization of mouse Rab32 by mRNA and protein expression analysis

Rab proteins, a subfamily of the ras superfamily, are low molecular weight GTPases involved in the regulation of intracellular vesicular transport. Cloning of human RAB32 was recently described. Presently, we report the cloning and characterization of the mouse homologue of Rab32. We show that murine Rab32 exhibits a ubiquitous expression pattern, with tissue-specific variation in expression level. Three cell types with highly specialized organelles, melanocytes, platelets and mast cells, exhibit relatively high level of Rab32. We show that in murine amelanotic in vitro transformed melanocytes as well as in human amelanotic metastatic melanoma cell lines, the expression of Rab32 is markedly reduced or absent, in parallel with the loss of expression of two key enzymes for the production of melanin, tyrosinase and Tyrp1. Therefore, in both mouse and human systems, the expression of Rab32 correlates with the expression of genes involved in pigment production. However, in melanoma samples, amelanotic due to a mutation in the tyrosinase gene, the expression of Rab32 remains at levels comparable to those observed in pigmented melanoma samples. Finally, we observed co-localization of Rab32 and the melanosomal proteins, Tyrp1 and Dct, indicating an association of Rab32 with melanosomes. Based on these data, we propose the inclusion of Rab32 to the so-called melanocyte/platelet family of Rab proteins.     

Vps9 Family Protein Muk1 Is the Second Rab5 Guanosine Nucleotide Exchange Factor in Budding Yeast

VPS9 domains can act as guanosine nucleotide exchange factors (GEFs) against small G proteins of the Rab5 family. Saccharomyces cerevisiae vps9Δ mutants have trafficking defects considerably less severe than multiple deletions of the three cognate Rab5 paralogs (Vps21, Ypt52, and Ypt53). Here, we show that Muk1, which also contains a VPS9 domain, acts as a second GEF against Vps21, Ypt52, and Ypt53. Muk1 is partially redundant with Vps9 in vivo, with vps9Δ muk1Δ double mutant cells displaying hypersensitivity to temperature and ionic stress, as well as profound impairments in endocytic and Golgi endosome trafficking, including defects in sorting through the multivesicular body. Cells lacking both Vps9 and Muk1 closely phenocopy double and triple knock-out strains lacking Rab5 paralogs. Microscopy and overexpression experiments demonstrate that Vps9 and Muk1 have distinct localization determinants. These experiments establish Muk1 as the second Rab5 GEF in budding yeast.     

A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery

The compartmental organization of eukaryotic cells is?maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the?cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface.     

Vesicle Associated Membrane Protein 8 (VAMP8)-mediated Zymogen Granule Exocytosis Is Dependent on Endosomal Trafficking via the Constitutive-Like Secretory Pathway

Acinar cell zymogen granules (ZG) express 2 isoforms of the vesicle-associated membrane protein family (VAMP2 and -8) thought to regulate exocytosis. Expression of tetanus toxin to cleave VAMP2 in VAMP8 knock-out (−/−) acini confirmed that VAMP2 and -8 are the primary VAMPs for regulated exocytosis, each contributing ∼50% of the response. Analysis of VAMP8^−/− acini indicated that although stimulated secretion was significantly reduced, a compensatory increase in constitutive secretion maintained total secretion equivalent to wild type (WT). Using a perifusion system to follow secretion over time revealed VAMP2 mediates an early rapid phase peaking and falling within 2–3 min, whereas VAMP8 controls a second prolonged phase that peaks at 4 min and slowly declines over 20 min to support the protracted secretory response. VAMP8^−/− acini show increased expression of the endosomal proteins Ti-VAMP7 (2-fold) and Rab11a (4-fold) and their redistribution from endosomes to ZGs. Expression of GDP-trapped Rab11a-S25N inhibited secretion exclusively from the VAMP8 but not the VAMP2 pathway. VAMP8^−/− acini also showed a >90% decrease in the early endosomal proteins Rab5/D52/EEA1, which control anterograde trafficking in the constitutive-like secretory pathway. In WT acini, short term (14–16 h) culture also results in a >90% decrease in Rab5/D52/EEA1 and a complete loss of the VAMP8 pathway, whereas VAMP2-secretion remains intact. Remarkably, rescue of Rab5/D52/EEA1 expression restored the VAMP8 pathway. Expressed D52 shows extensive colocalization with Rab11a and VAMP8 and partially copurifies with ZG fractions. These results indicate that robust trafficking within the constitutive-like secretory pathway is required for VAMP8- but not VAMP2-mediated ZG exocytosis.     

Vrl1 relies on its VPS9‐domain to play a role in autophagy in Saccharomyces cerevisiae

Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9‐domain‐containing protein, V AR P‐l ike 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9‐domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9‐domain‐containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9‐domain‐containing proteins, Vps9, Muk1, and Vrl1, all co‐localized with Atg8 on autophagosomes in cells blocked in any late step of starvation‐induced autophagy, with Vrl1 most often co‐localizing with Atg8. A small portion (<25%) of these VPS9‐domain‐containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy‐dependent and ‐independent routes.     

BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.     

Expression and characterization of Rab38, a new member of the Rab small G protein family

Rab38 is a new member of the Rab small G protein family that regulates intracellular vesicle trafficking. Rab38 is expressed in melanocytes and it has been clarified that a point mutation in the postulated GTP-binding domain of Rab38 is the gene responsible for oculocutaneous albinism in chocolate mice. However, basic information regarding recombinant protein production, intracellular location, and tissue-specific expression pattern has not yet been reported. We produced recombinant Rab38 using a baculovirus/insect cell-protein expression system. A combination of Triton X-114 phase separation and nickel-affinity chromatography yielded exclusively prenylated Rab38 that bound [alpha-32P]-GTP. The mRNA and the native protein were expressed in a tissue-specific manner, e.g., in the lung, skin, stomach, liver, and kidney. Freshly isolated rat alveolar type II cells were highly positive for the mRNA signal, but the signal was rapidly lost over time. Immunofluorescence staining demonstrated that expressed GST-tagged Rab38 was mainly co-localized with endoplasmic reticulum-resident protein and also partly with intermittent vesicles between the endoplasmic reticulum and the Golgi complex. These results indicate that Rab38 is expressed non-ubiquitously in specific tissues and regulates early vesicle transport relating to the endoplasmic reticulum, and hence suggest that Rab38 abnormality may cause multiple organ diseases as well as oculocutaneous albinism.     

Starvation‐induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome–lysosome fusion

Autophagy, the process for recycling cytoplasm in the lysosome, depends on membrane trafficking. We previously identified Drosophila Sbf as a Rab21 guanine nucleotide exchange factor (GEF) that acts with Rab21 in endosomal trafficking. Here, we show that Sbf/MTMR13 and Rab21 have conserved functions required for starvation‐induced autophagy. Depletion of Sbf/MTMR13 or Rab21 blocked endolysosomal trafficking of VAMP8, a SNARE required for autophagosome–lysosome fusion. We show that starvation induces Sbf/MTMR13 GEF and RAB21 activity, as well as their induced binding to VAMP8 (or closest Drosophila homolog, Vamp7). MTMR13 is required for RAB21 activation, VAMP8 interaction and VAMP8 endolysosomal trafficking, defining a novel GEF‐Rab‐effector pathway. These results identify starvation‐responsive endosomal regulators and trafficking that tunes membrane demands with changing autophagy status.     

Structural basis for Rab GTPase activation by VPS9 domain exchange factors

RABEX-5 and other exchange factors with VPS9 domains regulate endocytic trafficking through activation of the Rab family GTPases RAB5, RAB21 and RAB22. Here we report the crystal structure of the RABEX-5 catalytic core in complex with nucleotide-free RAB21, a key intermediate in the exchange reaction pathway. The structure reveals how VPS9 domain exchange factors recognize Rab GTPase substrates, accelerate GDP release and stabilize the nucleotide-free conformation. We further identify an autoinhibitory element in a predicted amphipathic helix located near the C terminus of the VPS9 domain. The autoinhibitory element overlaps with the binding site for the multivalent effector RABAPTIN-5 and potently suppresses the exchange activity of RABEX-5. Autoinhibition can be partially reversed by mutation of conserved residues on the nonpolar face of the predicted amphipathic helix or by assembly of the complex with RABAPTIN-5.     

EPI64 protein functions as a physiological GTPase-activating protein for Rab27 protein and regulates amylase release in rat parotid acinar cells

Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.     

The Rab Interacting Lysosomal Protein (RILP) Homology Domain Functions as a Novel Effector Domain for Small GTPase Rab36: Rab36 REGULATES RETROGRADE MELANOSOME TRANSPORT IN MELANOCYTES

Small GTPase Rab functions as a molecular switch that drives membrane trafficking through specific interaction with its effector molecule. Thus, identification of its specific effector domain is crucial to revealing the molecular mechanism that underlies Rab-mediated membrane trafficking. Because of the large numbers of Rab isoforms in higher eukaryotes, however, the effector domains of most of the vertebrate- or mammalian-specific Rabs have yet to be determined. In this study we screened for effector molecules of Rab36, a previously uncharacterized Rab isoform that is largely conserved in vertebrates, and we succeeded in identifying nine Rab36-binding proteins, including RILP (Rab interacting lysosomal protein) family members. Sequence comparison revealed that five of nine Rab36-binding proteins, i.e. RILP, RILP-L1, RILP-L2, and JIP3/4, contain a conserved coiled-coil domain. We identified the coiled-coil domain as a RILP homology domain (RHD) and characterized it as a common Rab36-binding site. Site-directed mutagenesis of the RHD of RILP revealed the different contributions by amino acids in the RHD to binding activity toward Rab7 and Rab36. Expression of RILP in melanocytes, but not expression of its Rab36 binding-deficient mutants, induced perinuclear aggregation of melanosomes, and this effect was clearly attenuated by knockdown of endogenous Rab36 protein. Moreover, knockdown of Rab36 in Rab27A-deficient melanocytes, which normally exhibit perinuclear melanosome aggregation because of increased retrograde melanosome transport activity, caused dispersion of melanosomes from the perinucleus to the cell periphery, but knockdown of Rab7 did not. Our findings indicated that Rab36 mediates retrograde melanosome transport in melanocytes through interaction with RILP.