Rab2 requires PKC iota/lambda to recruit beta-COP for vesicle formation

Traffic. 2000 Sep;1(9):702-12. doi: 10.1034/j.1600-0854.2000.010903.x.

Abstract

The small GTPase Rab2 initiates the recruitment of soluble components necessary for protein sorting and recycling from pre-Golgi intermediates. Our previous studies showed that Rab2 required protein kinase C (PKC) or a PKC-like protein to recruit beta-COP to membrane (Tisdale EJ, Jackson M. Rab2 protein enhances coatomer recruitment to pre-Golgi intermediates. J Biol Chem 1998;273: 17269-17277). We investigated the role of PKC in Rab2 function by first determining the active isoform that associates with membranes used in our assay. Western blot analysis detected three isoforms: PKC alpha, gamma and iota/lambda. A quantitative binding assay was used to measure recruitment of these kinases when incubated with Rab2. Only PKC iota/lambda translocated to membrane in a dose-dependent manner. Microsomes treated with anti-PKC iota/lambda lost the ability to bind beta-COP, suggesting that Rab2 requires PKC iota/lambda for beta-COP recruitment. The recruitment of beta-COP to membranes is not regulated by PKC iota/lambda kinase activity. However, PKC iota/lambda activity was necessary for Rab2-mediated vesicle budding. We found that the addition of either a kinase-deficient PKC iota/lambda mutant or atypical PKC pseudosubstrate peptide to the binding assay drastically reduced vesicle formation. These data suggest that Rab2 causes translocation of PKC iota/lambda to vesicular tubular clusters (VTCs), which promotes the recruitment of COPI to generate retrograde-transport vesicles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured / metabolism
  • Coatomer Protein / metabolism*
  • Cytosol / metabolism
  • Golgi Apparatus / metabolism
  • Intracellular Membranes / metabolism
  • Microsomes / metabolism
  • Protein Isoforms / metabolism*
  • Protein Kinase C / metabolism*
  • Protein Transport / physiology*
  • Transport Vesicles / metabolism*
  • rab2 GTP-Binding Protein / metabolism*

Substances

  • Coatomer Protein
  • Protein Isoforms
  • Protein Kinase C
  • rab2 GTP-Binding Protein