Abstract
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.
Highlights
Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules
Molecular Cloning of Full-length Rab-binding Proteins by PCR— Because most of the clones we identified by yeast two-hybrid screening contained a fragment of Rab-binding proteins, the full-length cDNA of some of these proteins (i.e. AI507611/ Golgi-associated Rab2B interactor (GARI),1 OTTMUSG00000005491/ GARI-like3 (GARI-L3), nuclear distribution gene E homolog 1 (Nde1), WD repeat domain 38 (Wdr38), inositol-polyphosphate 5-phosphatase E (INPP5E), 2900002H16Rik, C-terminal binding protein 1 (CtBP1), and component of oligomeric Golgi complex 4 (Cog4)) was obtained by PCR from Marathon-Ready adult mouse testis cDNA using the following oligonucleotides with a restriction enzyme site or a stop codon as described previously [15]: 5Ј-GGATCCATGAGTAAAATTAGGGGCCT-3Ј (GARI Met primer, sense) and 5Ј-TTAGGGTCCCAACAAGTTCT-3Ј (GARI stop primer, antisense); 5Ј-GGATCCATGCCTGGGATGAAGAG
Other Proteins That Bind Multiple Rab Isoforms—In addition to MICALs/MICAL-Ls and OCRL/inositol-polyphosphate 5-phosphatase B (INPP5B), we identified four novel Rab-binding proteins that exhibit broad Rab binding specificity (Smchd1, 2900002H16Rik, AKap10, and 4930573I19Rik) (Fig. 4A and Table I)
Summary
Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. 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. Approximately two-thirds (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms, whereas the others recognized only a single Rab isoform (or members of a single Rab subfamily). Based on these findings, we discuss the specificity and diversity of Rab-effector interactions in membrane trafficking
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