Genome-wide Investigation of the Rab Binding Activity of RUN Domains: Development of a Novel Tool That Specifically Traps GTP-Rab35

Cell Struct Funct. 2011;36(2):155-70. doi: 10.1247/csf.11001. Epub 2011 Jul 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Carrier Proteins
  • Nuclear Proteins
  • Protein Isoforms
  • RUFY3 protein, human
  • RUSC2 protein, human
  • Recombinant Proteins
  • Rufy2 protein, mouse
  • Guanosine Triphosphate
  • rab GTP-Binding Proteins
  • rab5 GTP-Binding Proteins