Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates

Ellen J. Tisdale, Jing Wang, Robert B. Silver, Cristina R. Artalejo

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

The small GTPase Rab2 requires atypical protein kinase C ι/λ (PKCι/λ) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCι/λ in the early secretory pathway. A peptide corresponding to the unique PKCι/λ pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCι/λ, β-COP, and p53/p58. Because Rab2, β-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters, VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCι/λ (W274K). Both the pseudosubstrate peptide and kinase-dead PKCι/λ in tandem with Rab2 caused sustained membrane association of PKCι/λ, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCι/λ was reversed by PKCι/λ wild type. These combined results indicate that PKCι/λ is essential for protein transport in the early secretory pathway and suggest that PKCι/λ kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.

Original languageEnglish (US)
Pages (from-to)38015-38021
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number39
DOIs
StatePublished - Sep 26 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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