Study of the Plant COPII Vesicle Coat Subunits by Functional Complementation of Yeast Saccharomyces cerevisiae Mutants

Johan-Owen De Craene,Mari Carmen Herranz,Chantal Fitterer,Corinne Schmitt-Keichinger,Fanny Courte,Bruno Rinaldi,Sylvie Friant,Christophe Ritzenthaler,Hong Wanjin

doi:10.1371/journal.pone.0090072

Abstract

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23–Sec24 heterodimer following the subsequent recruitment of the Sec13–Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Highlights

The formation and loading of endoplasmic reticulum ERderived vesicles is a highly conserved function in eukaryotic cells and requires a vesicular coat termed the coat protein complex II (COPII) that was first identified in yeast Saccharomyces cerevisiae [1,2]
The numerous COPII constituents found in A. thaliana were analyzed by heterologous expression in S. cerevisiae and tested for their ability to complement yeast loss-of-function mutations
This first required the identification of all A. thaliana COPII proteins by BLAST analysis using the corresponding yeast proteins as bait

Summary

Introduction

The formation and loading of endoplasmic reticulum ERderived vesicles is a highly conserved function in eukaryotic cells and requires a vesicular coat termed the coat protein complex II (COPII) that was first identified in yeast Saccharomyces cerevisiae [1,2]. The COPII coat is constituted of five cytoplasmic proteins Sar, Sec, Sec, Sec and Sec and one ER resident transmembrane protein Sec. The COPII coat is constituted of five cytoplasmic proteins Sar, Sec, Sec, Sec and Sec and one ER resident transmembrane protein Sec12 Together they participate to the stepwise COPII vesicle assembly process [3]. Activated Sar interacts with the Sec23–Sec heterodimeric complex, which recruits the cargos and forms a subregion of the ER membrane enriched in COPII proteins. This results in the recruitment of the Sec13–Sec heterotetramer by the Sec23–Sec complex and induces ER membrane bending [4]. After vesicle formation and prior to vesicle fusion with the Golgi, the vesicle uncoats after GTP hydrolysis by Sar stimulated by the GTPase activating protein domain of Sec23 [5]

Methods

Results

Conclusion