Rab2 Requires PKCι/Λ to Recruit β‐COP for Vesicle Formation

Open Access
Publisher Website
Google Scholar
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 β‐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: PKCα, Γ and ι/Λ. A quantitative binding assay was used to measure recruitment of these kinases when incubated with Rab2. Only PKCι/Λ translocated to membrane in a dose‐dependent manner. Microsomes treated with anti‐PKCι/Λ lost the ability to bind β‐COP, suggesting that Rab2 requires PKCι/Λ for β‐COP recruitment. The recruitment of β‐COP to membranes is not regulated by PKCι/Λ kinase activity. However, PKCι/Λ kinase activity was necessary for Rab2‐mediated vesicle budding. We found that the addition of either a kinase‐deficient PKCι/Λ mutant or atypical PKC pseudosubstrate peptide to the binding assay drastically reduced vesicle formation. These data suggest that Rab2 causes translocation of PKCι/Λ to v esicular t ubular c lusters (VTCs), which promotes the recruitment of COPI to generate retrograde‐transport vesicles.