Exocyst Sec10 is involved in basolateral protein translation and translocation in the endoplasmic reticulum

Abstract

Background: Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly made proteins are translocated into or across the RER membrane consists of three main subunits: Sec61α, -β, and -γ. Sec61β facilitates translocation, and we and others have shown that the highly conserved eight-protein exocyst complex interacts with Sec61β. We have also shown that the exocyst is involved in basolateral, not apical, protein synthesis and delivery. Recently, however, exocyst involvement in apical protein delivery has been reported. Furthermore, we have shown that the exocyst is necessary for formation of primary cilia, organelles found on the apical surface. Methods: GST pulldown was performed on lysate of renal tubule cells to investigate biochemical interactions. Cell-free assays consisting of cell-free extracts from rabbit reticulocytes, pancreatic endoplasmic reticulum (ER) microsomal membranes, transcripts of cDNA from apical and basolateral proteins, ATP/GTP, amino acids, and 35S-methionine for protein detection were used to investigate the role of the exocyst in synthesis of polarized proteins. P32-orthophosphate and immunoprecipitation with antibody against Sec61β was used to investigate Sec61β phosphorylation in exocyst Sec10-overexpressing cells. Results: Sec10 biochemically interacts with Sec61β using GST pulldown. Using cell-free assays, there is enhanced exocyst recruitment to endoplasmic reticulum membranes following exocyst depletion and basolateral G protein of vesicular stomatitis virus protein translation, compared to apical hemagglutinin of influenza virus protein translation. Finally, Sec10 overexpression increases Sec61β phosphorylation. Conclusion: These data confirm that the exocyst is preferentially involved in basolateral protein translation and translocation, and may well act through the phosphorylation of Sec61β. Copyright © 2012 S. Karger AG, Basel.