Eiko Kanno

Golgi-resident Small GTPase Rab33B Interacts with Atg16L and Modulates Autophagosome Formation

Macroautophagy is a mechanism of degradation of cytoplasmic components in all eukaryotic cells. In macroautophagy, cytoplasmic components are wrapped by double-membrane structures called autophagosomes, whose formation involves unique membrane dynamics, i.e., de novo formation of a double-membrane sac called the isolation membrane and its elongation. However, the precise regulatory mechanism of isolation membrane formation and elongation remains unknown. In this study, we showed that Golgi-resident small GTPase Rab33B (and Rab33A) specifically interacts with Atg16L, an essential factor in isolation membrane formation, in a guanosine triphosphate-dependent manner. Expression of a GTPase-deficient mutant Rab33B (Rab33B-Q92L) induced the lipidation of LC3, which is an essential process in autophagosome formation, even under nutrient-rich conditions, and attenuated macroautophagy, as judged by the degradation of p62/sequestosome 1. In addition, overexpression of the Rab33B binding domain of Atg16L suppressed autophagosome formation. Our findings suggest that Rab33 modulates autophagosome formation through interaction with Atg16L.

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.

Conserved N-terminal Cysteine Motif Is Essential for Homo- and Heterodimer Formation of Synaptotagmins III, V, VI, and X

The synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca2+ sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267–12273) forms β-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts III, V, VI, and X. We also show that native Syt III from mouse brain forms a β-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca2+ sensor for neurotransmitter release.

Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity.

It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase‐activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab‐GAP activity. In this study we established panels of the GTP‐fixed form of 60 different Rabs constructed in pGAD‐C1, a yeast two‐hybrid bait vector. We also constructed a yeast two‐hybrid prey vector (pGBDU‐C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP‐fixed Rabs and 40 TBC proteins by yeast two‐hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5‐like with Rab10). Measurement of in vitro Rab‐GAP activity revealed that OATL1 and Evi5‐like actually possess significant Rab2A‐ and Rab10‐GAP activity, respectively, but that FLJ12085 do not display Rab5A‐GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab‐GAP domains, but that there is little correlation between the Rab‐binding activity and Rab‐GAP activity of other TBC proteins.

OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation

The GAP activity of OATL1, which is recruited to autophagosomes by Atg8, regulates autophagosome–lysosome fusion.

Myosin Vb Is Required for Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator in Rab11a-specific Apical Recycling Endosomes in Polarized Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl- channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and ΔF508-CFTR in the apical membrane and decreased CFTR-mediated Cl- secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.

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.

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.

Rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27A in PC12 cells

Rabphilin and Noc2 were originally described as Rab3A effector proteins involved in the regulation of secretory vesicle exocytosis, however, recently both proteins have been shown to bind Rab27A in vitro in preference to Rab3A (Fukuda, M. (2003) J. Biol. Chem. 278, 15373-15380), suggesting that Rab3A is not their major ligand in vivo. In the present study we showed by means of deletion and mutation analyses that rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27A, not with Rab3A, in PC12 cells. Rab3A binding-defective mutants of rabphilin(E50A) and Noc2(E51A) were still localized in the distal portion of the neurites (where dense-core vesicles had accumulated) in nerve growth factor-differentiated PC12 cells, the same as the wild-type proteins, whereas Rab27A binding-defective mutants of rabphilin(E50A/I54A) and Noc2(E51A/I55A) were present throughout the cytosol. We further showed that expression of the wild-type or the E50A mutant of rabphilin-RBD, but not the E50A/I54A mutant of rabphilin-RBD, significantly inhibited high KCl-dependent neuropeptide Y secretion by PC12 cells. We also found that rabphilin and its binding partner, Rab27 have been highly conserved during evolution (from nematoda to humans) and that Caenorhabditis elegans and Drosophila rabphilin (ce/dm-rabphilin) specifically interact with ce/dm-Rab27, but not with ce/dm-Rab3 or ce/dm-Rab8, suggesting that rabphilin functions as a Rab27 effector across phylogeny. Based on these findings, we propose that the N-terminal Rab binding domain of rabphilin and Noc2 be referred to as “RBD27 (Rab binding domain for Rab27)”, the same as the synaptotagmin-like protein homology domain (SHD) of Slac2-a/melanophilin.

Slp4-a/granuphilin-a regulates dense-core vesicle exocytosis in PC12 cells.

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic β-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999)J. Biol. Chem. 274, 28542–28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitroand in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212–9218). How Slp4-a·Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a·Rab8 and Slp4-a·Rab27A complexes, but not Slp4-a·Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A.