Mammalian Sec16/p250 Plays a Role in Membrane Traffic from the Endoplasmic Reticulum

Akiko Shiga,Takayuki Iinuma,Katsuko Tani,Mitsuo Tagaya,Nagisa Arimitsu,Matthew B. O'Brien,Koji Nakamoto,Meir Aridor

doi:10.1074/jbc.m611237200

Abstract

Coat protein complex II (COPII)-coated vesicles/carriers, which mediate export of proteins from the endoplasmic reticulum (ER), are formed at special ER subdomains in mammals, termed ER exit sites or transitional ER. The COPII coat consists of a small GTPase, Sar1, and two protein complexes, Sec23-Sec24 and Sec13-Sec31. Sec23-Sec24 and Sec13-Sec31 appear to constitute the inner and the outermost layers of the COPII coat, respectively. We previously isolated two mammalian proteins (p125 and p250) that bind to Sec23. p125 was found to be a mammalian-specific, phospholipase A(1)-like protein that participates in the organization of ER exit sites. Here we show that p250 is encoded by the KIAA0310 clone and has sequence similarity to yeast Sec16 protein. Although KIAA0310p was found to be localized at ER exit sites, subcellular fractionation revealed its predominant presence in the cytosol. Cytosolic KIAA0310p was recruited to ER membranes in a manner dependent on Sar1. Depletion of KIAA0310p mildly caused disorganization of ER exit sites and delayed protein transport from the ER, suggesting its implication in membrane traffic out of the ER. Overexpression of KIAA0310p affected ER exit sites in a manner different from that of p125. Binding experiments suggested that KIAA0310p interacts with both the inner and the outermost layer coat complexes, whereas p125 binds principally to the inner layer complex. Our results suggest that KIAA0310p, a mammalian homologue of yeast Sec16, builds up ER exit sites in cooperation with p125 and plays a role in membrane traffic from the ER.

Highlights

complex II (COPII)-coated vesicles, which are generated at endoplasmic reticulum (ER) subdomains, known as ER exit sites (ERES), in many eukaryotes [3, 4]
We previously isolated two mammalian Sec23-interacting proteins (p125 and p250) by using glutathione beads coupled to glutathione S-transferase (GST)-mouse Sec23A [30]. p125, which appears to be only expressed in mammals, contains an N-terminal Pro-rich region responsible for the interaction with Sec23 [31], as well as central and C-terminal regions, which coprotein; PA-PLA1, phosphatidic acid-preferring phospholipase A1; MS, mass spectrometry; MALDI-QqTOF, matrix-assisted laser desorption/ionization-quadrupole time of flight
To confirm that p250 is KIAA0310p, we raised a polyclonal antibody against a mixture of two peptides derived from the sequence of human KIAA0310p and examined whether endogenous KIAA0310p is pulled down with GST-Sec23A expressed in 293T cells

Summary

Introduction

COPII-coated vesicles, which are generated at ER subdomains, known as ER exit sites (ERES), in many eukaryotes [3, 4]. Studies by Glick and colleagues [26, 27] have shown that a tightly ER-bound peripheral membrane protein, Sec16, is required for the organization of ERES in P. pastoris. KIAA0310p, which shows sequence similarity to yeast Sec16 protein, was found to be localized at ERES, and its depletion induced their disorganization.

Results

Conclusion