Involvement of the Penta-EF-Hand Protein Pef1p in the Ca2+-Dependent Regulation of COPII Subunit Assembly in Saccharomyces cerevisiae

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan.
PLoS ONE (Impact Factor: 3.23). 07/2012; 7(7):e40765. DOI: 10.1371/journal.pone.0040765
Source: PubMed


Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca(2+)-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca(2+)-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca(2+) dependence for Sec13/31p association; only the Ca(2+)-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca(2+)-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.

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    • "It is possible that ALG-2 has evolved in higher eukaryotes in order to regulate sorting of proteins to ERGIC. The PEF-family yeast homologue, Pef1p was recently reported to bind to the Sec13/31p in a Ca2+-independent manner [28]. We were not able to detect binding between Sec31A and the mammalian homologues of Pef1p, Peflin (not shown). "
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    ABSTRACT: Coated vesicles mediate the traffic of secretory and membrane cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The coat protein complex (COPII) involved in vesicle budding is constituted by a GTPase, Sar1, the inner coat components of Sec23/Sec24 and the components of the outer coat Sec13/Sec31A. The Ca(2+)-binding protein ALG-2 was recently identified as a Sec31A binding partner and a possible link to Ca(2+) regulation of COPII vesicle budding. Here we show that ALG-2/Ca(2+) is capable of attenuating vesicle budding in vitro through interaction with an ALG-2 binding domain in the proline rich region of Sec31A. Binding of ALG-2 to Sec31A and inhibition of COPII vesicle budding is furthermore dependent on an intact Ca(2+)-binding site at EF-hand 1 of ALG-2. ALG-2 increased recruitment of COPII proteins Sec23/24 and Sec13/31A to artificial liposomes and was capable of mediating binding of Sec13/31A to Sec23. These results introduce a regulatory role for ALG-2/Ca(2+) in COPII tethering and vesicle budding.
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    • "In these distinct zones of the ER, a set of cytoplasmic proteins, collectively known as the COPII coat, generates COPII vesicles through a sequence of events under the control of multiple regulatory mechanisms (Aridor and Balch, 2000; Lee and Linstedt, 2000; Blumental-Perry et al., 2006; Yamasaki et al., 2006; Higashio et al., 2008; Rismanchi et al., 2009; Kodera et al., 2011; Yorimitsu and Sato, 2012; Yoshibori et al., 2012). The COPII coat is responsible for the direct or indirect capture of cargo proteins and for the physical deformation of the ER membrane that drives the COPII vesicle formation. "
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