VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking

J Cell Biol. 2016 Aug 15;214(4):445-58. doi: 10.1083/jcb.201604061. Epub 2016 Aug 8.

Abstract

Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cytosol / metabolism*
  • Dogs
  • GTP Phosphohydrolases / metabolism*
  • Gene Silencing
  • Green Fluorescent Proteins / metabolism
  • Guanosine Triphosphate / metabolism
  • HEK293 Cells
  • Humans
  • Jurkat Cells
  • Lipoylation
  • Madin Darby Canine Kidney Cells
  • Melanoma / metabolism
  • Membrane Proteins / metabolism*
  • Molecular Weight
  • Mutant Proteins / metabolism
  • Protein Binding
  • Protein Prenylation*
  • Protein Transport
  • Subcellular Fractions / metabolism
  • Vesicular Transport Proteins / metabolism*

Substances

  • Membrane Proteins
  • Mutant Proteins
  • VPS35 protein, human
  • Vesicular Transport Proteins
  • Green Fluorescent Proteins
  • Guanosine Triphosphate
  • GTP Phosphohydrolases
  • NRAS protein, human