RRC ID 48876
Author Fukuda M, Kobayashi H, Ishibashi K, Ohbayashi N.
Title Genome-wide investigation of the Rab binding activity of RUN domains: development of a novel tool that specifically traps GTP-Rab35.
Journal Cell Struct Funct
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 domain-containing 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.
Volume 36(2)
Pages 155-70
Published 2011-1-1
DOI 10.1247/csf.11001
PII JST.JSTAGE/csf/11001
PMID 21737958
MeSH Animals Carrier Proteins / chemistry Carrier Proteins / metabolism Genome* Genome-Wide Association Study / methods Guanosine Triphosphate / metabolism* Humans Mice Neurites / metabolism Nuclear Proteins / chemistry Nuclear Proteins / metabolism PC12 Cells Protein Binding Protein Isoforms / chemistry Protein Isoforms / metabolism Protein Structure, Tertiary Rats Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / metabolism rab GTP-Binding Proteins / chemistry rab GTP-Binding Proteins / genetics rab GTP-Binding Proteins / metabolism* rab5 GTP-Binding Proteins / chemistry rab5 GTP-Binding Proteins / metabolism
IF 2.429
Times Cited 47
DNA material pEGFP-C1-RBD35 (RDB15105) pGEX-4T-3-RBD35 (RDB15106) pEGFP-C1-mouse DENND1A (RDB15289) pMRX-puro-EGFP-DENND1A (RDB17952)