Koutaro Ishibashi

Large Scale Screening for Novel Rab Effectors Reveals Unexpected Broad Rab Binding Specificity

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1–30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast two-hybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.

Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

After bacterial invasion, ubiquitin is conjugated to host endosomal proteins and recognized by the autophagic machinery independent of LC3.

Comprehensive screening for novel rab-binding proteins by GST pull-down assay using 60 different mammalian Rabs.

The Rab family belongs to the Ras‐like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S‐transferase (GST) pull‐down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab‐binding proteins we identified, mKIAA1055/TBC1D2B (Rab22‐binding protein), GAPCenA/TBC1D11 (Rab36‐binding protein) and centaurin β2/ACAP2 (Rab35‐binding protein), are GTPase‐activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab–GAP (Tre‐2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin β2 binds GTP‐Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin β2 did not exhibit any Rab35–GAP activity in vitro, the Rab35‐binding ANKR domain of centaurin β2 was found to be required for its plasma membrane localization and regulation of Rab35‐dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.

Structure-Function Analysis of VPS9-Ankyrin-repeat Protein (Varp) in the Trafficking of Tyrosinase-related Protein 1 in Melanocytes

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking.

Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity

Atg16L1, a protein essential for autophagy, is localized on dense-core vesicles in PC12 cells, and knockdown of Atg16L1 inhibits hormone secretion independently of autophagy. In addition, Atg16L1 interacts with the small GTPase Rab33A, and this interaction is required for the dense-core vesicle localization of Atg16L1.

Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12–5-16L2 complex

A large protein complex consisting of Atg5, Atg12 and Atg16L1 has recently been shown to be essential for the elongation of isolation membranes (also called phagophores) during mammalian autophagy. However, the precise function and regulation of the Atg12–5-16L1 complex has largely remained unknown. In this study we identified a novel isoform of mammalian Atg16L, termed Atg16L2, that consists of the same domain structures as Atg16L1. Biochemical analysis revealed that Atg16L2 interacts with Atg5 and self-oligomerizes to form an ~800-kDa complex, the same as Atg16L1 does. A subcellular distribution analysis indicated that, despite forming the Atg12–5-16L2 complex, Atg16L2 is not recruited to phagophores and is mostly present in the cytosol. The results also showed that Atg16L2 is unable to compensate for the function of Atg16L1 in autophagosome formation, and knockdown of endogenous Atg16L2 did not affect autophagosome formation, indicating that Atg16L2 does not possess the ability to mediate canonical autophagy. Moreover, a chimeric analysis between Atg16L1 and Atg16L2 revealed that their difference in function in regard to autophagy is entirely attributable to the difference between their middle regions that contain a coiled-coil domain. Based on the above findings, we propose that formation of the Atg12–5-16L complex is necessary but insufficient to mediate mammalian autophagy and that an additional function of the middle region (especially around amino acid residues 229–242) of Atg16L1 (e.g., interaction with an unidentified binding partner on phagophores) is required for autophagosome formation.

Identification and characterization of a novel Tre-2/Bub2/Cdc16 (TBC) protein that possesses Rab3A-GAP activity.

The Tre‐2/Bub2/Cdc16 (TBC) domain is a conserved protein motif that consists of approximately 200 amino acids and is thought to function as a specific Rab‐GAP domain. Although more than 40 distinct TBC domain‐containing proteins have been identified in humans, the GAP activity and specificity of most TBC proteins have never been determined. In this study we developed a novel method of screening for Rab3A‐GAP and identified two TBC proteins (FLJ13130 and RN‐tre) whose expression in PC12 cells was associated with exclusion of endogenous Rab3A molecules from dense‐core vesicles. As expression of RN‐tre caused fragmentation of the Golgi, which presumably resulted in the loss of dense‐core vesicles themselves, we further characterized FLJ13130 as a candidate Rab3A‐GAP. The results showed that expression of FLJ13130, but not of its catalytically inactive R134K mutant, greatly reduced the amount of GTP‐Rab3A in living cells and promoted the GTPase activity of Rab3A in vitro. Unexpectedly, however, FLJ13130 also promoted the GTPase activity of Rab22A, Rab27A, and Rab35, but not of Rab2A or Rab6A. Based on these results, we propose that FLJ13130 is a novel type of Rab‐GAP that exhibits broad GAP specificity and inactivates several distinct Rab isoforms, including Rab3A, just near the plasma membrane.

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.

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

Human Griscelli syndrome type 2 (GS‐2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2‐a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS‐2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2‐a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2‐a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2‐a, Slp4‐a/granuphilin‐a, and Munc13‐4. The Rab27A(K22R) mutant normally binds Munc13‐4, but not Slp2‐a or Slp4‐a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2‐a and Munc13‐4 but normally binds Slp4‐a.

Atg16L1 Protein Regulates Hormone Secretion Independent of Autophagy

Atg16L1 is an essential factor for canonical autophagy, a conserved bulk degradation system in all eukaryotes. Atg16L1 forms a complex with Atg12-conjugated Atg5, i.e., an Atg16L1–5–12 complex, and promotes elongation of isolation membranes possibly by recruiting LC3 and by facilitating its lipidation. Because of its critical role in canonical autophagy, cells from Atg16L1-deficient mice exhibit complete loss of autophagosome formation. Interestingly, the intestinal Paneth cells of Atg16L1-deficient mice exhibit a secretion defect, but the mechanism by which Atg16L1 regulates the secretory pathway is poorly understood. We recently reported the finding that Atg16L1 localizes on hormone-containing dense-core vesicles in neuroendocrine PC12 cells independent of canonical autophagy and that small GTPase Rab33A recruits the Atg16L1–5–12 complex to dense-core vesicles. We also found that knockdown of Atg16L1 in PC12 cells caused a dramatic reduction in hormone secretion independent of the autophagic activity of the cells. Our findings indicate that, in addition to its role in autophagy, Atg16L1 (or the Atg16L1–5–12 complex) regulates hormone secretion from dense-core vesicles, most likely by acting as a Rab33A effector in particular cell types, including PC12 cells.