par-1, Atypical pkc, and PP2A/B55 sur-6 Are Implicated in the Regulation of Exocyst-Mediated Membrane Trafficking in Caenorhabditis elegans

• Published in: G3 : genes - genomes - genetics 2014-01, Vol.4 (1), p.173-183
• Main Authors: Jiu, Yaming, Hasygar, Kiran, Tang, Lois, Liu, Yanbo, Holmberg, Carina I, Bürglin, Thomas R, Hietakangas, Ville, Jäntti, Jussi
• Format: Article
• Language: English
• Subjects: Alleles; Animals; Animals, Genetically Modified; Caenorhabditis elegans - metabolism; Caenorhabditis elegans Proteins - antagonists & inhibitors; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Cell Membrane - metabolism; Exocytosis; Medicin och hälsovetenskap; Mutant Screen Report; Mutation; Phenotype; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - genetics; Protein Kinase C - metabolism; Protein Phosphatase 2 - antagonists & inhibitors; Protein Phosphatase 2 - genetics; Protein Phosphatase 2 - metabolism; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; RNA Interference; SNARE Proteins - genetics; SNARE Proteins - metabolism; Vesicular Transport Proteins - genetics; Vesicular Transport Proteins - metabolism
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1534/g3.113.006718

Abstract The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process.