Rab2 requires PKC iota/lambda to recruit beta-COP for vesicle formation.
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.
Tisdale
E J
EJ
Department of Pharmacology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201, USA. etisdale@med.wayne.edu
D000818:Animals; D002478:Cells, Cultured / Q000378:metabolism; D020756:Coatomer Protein / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D022161:Transport Vesicles / Q000378:metabolism*; D020693:rab2 GTP-Binding Protein / Q000378:metabolism*
D020756:Coatomer Protein; D020033:Protein Isoforms; D011493:Protein Kinase C; D020693:rab2 GTP-Binding Protein