Rab11-FIP2 Interaction With MYO5B Regulates Movement of Rab11a-containing Recycling Vesicles

Traffic. 2014 Mar;15(3):292-308. doi: 10.1111/tra.12146. Epub 2014 Jan 22.

Abstract

A tripartite association of Rab11a with both Rab11-FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11-FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11-FIP2 that caused loss of interaction with MYO5B in yeast two-hybrid assays as well as loss of interaction of Rab11-FIP2(129-356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full-length Rab11-FIP2 with MYO5B tail in HeLa cells. While EGFP-Rab11-FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP-Rab11-FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a-containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11-FIP2 association is perturbed by mutation or by Rab11-FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11-FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.

Keywords: MYO5B; Rab11; Rab11-FIP2; membrane recycling.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Dogs
  • Endosomes / metabolism*
  • HeLa Cells
  • Humans
  • Madin Darby Canine Kidney Cells
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Myosin Heavy Chains / genetics
  • Myosin Heavy Chains / metabolism*
  • Myosin Type V / genetics
  • Myosin Type V / metabolism*
  • Point Mutation
  • Protein Binding
  • Protein Transport
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Carrier Proteins
  • MYO5B protein, human
  • Membrane Proteins
  • Myosin Type V
  • rab11 protein
  • Myosin Heavy Chains
  • Rab11-FIP2 protein, human
  • rab GTP-Binding Proteins