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

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 twohybrid 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.

† These authors contributed equally to this work. ‡ The nucleotide sequences reported in this article have been submitted to the GenBank/EBI Data Bank with accession numbers AB490692-3.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 GTPaseactivating 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.

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 Rab32/38⅐Varp complex by performing Ala-based site-directed mutagenesis, and we dis- Pigmentation of mammalian hair and skin requires proper formation and transport of melanosomes, one of the lysosomerelated organelles that specifically synthesize and store melanin pigments, in melanocytes (reviewed in Refs. 1, 2). Defects in the formation and/or transport of melanosomes often cause pigmentary disorders, e.g. albinism and pigmentary dilution, in mammals (reviewed in Ref.3). The formation and transport of melanosomes involve a variety of intracellular membrane trafficking events, and several distinct Rab-type small GTPases, which are conserved membrane trafficking proteins in all eukaryotic cells (reviewed in Refs. 4 -6), have been shown to regulate the maturation and transport of melanosomes in mammalian epidermal melanocytes.The best characterized Rab isoform that is abundant on mature melanosomes in melanocytes is Rab27A (7-9). Rab27A regulates actin-based melanosome transport through interaction with its specific effector, Slac2-a/melanophilin (10 -12), and melanosome anchoring to the plasma membrane through interaction with another effector, Slp2-a (13). As a result, Rab27A deficiency causes human type 2 Griscelli syndrome, which is characterized by silvery hair (i.e. partial albinism) (reviewed in Ref. 14 and references therein). Two other Rab isoforms, Rab32 and Rab38, regulate an early step in melanogenesis, i.e. the transport of melanogenic enzymes to melanosomes (15-17). Actually, dysfunction of Rab38 causes the diluted coat color of chocolate mice, presumably because of impairment of the targeting of tyrosinase-related protein 1 (Tyrp1) to melanosomes (15). The discovery of the Rab32/38-specific binding protein Varp 4 (VPS9-ankyrin-repeat protein; official symbol in the National Center for Biotechnology Information is Ankrd27) has recently been reported (18,19), and as its name indicates, Varp protein contains an N-terminal VPS9 (vacuolar protein sorting 9) domain and C-terminal tandem ankyrin repeat domains (named ANKR1 and ANKR2). The Varp VPS9 domain possesses Rab21-GEF (guanine nucleotide exchange factor) activity (20), and the ANKR1 domain functions as a specific GTP-Rab32/38-binding site (18,19). Even more recently, VAMP7/TI-VAMP (vesicle-associated membrane protein) has been shown to interact with the region between the ANKR1 domain and the ANKR2 domain and t...

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.

ABSTRACT. The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids, and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site. However, since the Rab binding activity of most RUN domains has never been investigated, in this study we performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domaincontaining proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six of them interact with specific Rab isoforms with different Rab binding specificity, i.e., DENND5A/B with Rab6A/ B, PLEKHM2 with Rab1A, RUFY2/3 with Rab33, and RUSC2 with Rab1/Rab35/Rab41. We also identified the minimal functional Rab35-binding site of RUSC2 (amino acid residues 982-1199) and succeeded in developing a novel GTP-Rab35-specific trapper, which we named RBD35 (Rab-binding domain specific for Rab35). Recombinant RBD35 was found to trap GTP-Rab35 specifically both in vitro and in PC12 cells, and overexpression of fluorescently tagged RBD35 in PC12 cells strongly inhibited nerve growth factor-dependent neurite outgrowth.

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.

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.