Involvement of the Penta-EF-Hand Protein Pef1p in the
Ca2+-Dependent Regulation of COPII Subunit Assembly
in Saccharomyces cerevisiae
Mariko Yoshibori, Tomohiro Yorimitsu, Ken Sato*
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan
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 Ca2+-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 Ca2+-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the
membrane. However, Pef1p displayed reversed Ca2+dependence for Sec13/31p association; only the Ca2+-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 Ca2+-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.
Citation: Yoshibori M, Yorimitsu T, Sato K (2012) Involvement of the Penta-EF-Hand Protein Pef1p in the Ca2+-Dependent Regulation of COPII Subunit Assembly
in Saccharomyces cerevisiae. PLoS ONE 7(7): e40765. doi:10.1371/journal.pone.0040765
Editor: Wanjin Hong, Institute of Molecular and Cell Biology, Singapore
Received May 20, 2012; Accepted June 13, 2012; Published July 11, 2012
Copyright: ? 2012 Yoshibori et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant-in-aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS) (TY and KS), and in part by
the Targeted Proteins Research Program from MEXT:Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) (KS). The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Membrane trafficking is a fundamental mechanism for com-
munication between distinct membrane-bound organelles within a
cell. Transport vesicles are used for the delivery of a variety of
molecules from one compartment to another. In addition to
delivery of protein molecules, these transport vesicles allow
exchange of membrane lipids between organelles. Thus, size of
the secretory organelle might largely rely on the regulatory
mechanisms of vesicular transport that ensure a proper balance of
membrane input and output at each organelle. The endoplasmic
reticulum (ER)-to-cis-Golgi transport step is mediated by coat
protein complex II (COPII)-coated vesicles in the anterograde
direction, and by COPI-coated vesicles in the retrograde direction
, . Therefore, the net flux of membrane into and out of the
ER, for example, would be determined by the rate of vesicular
transport from the cis-Golgi, which contributes to input of vesicles,
and the rate of vesicular transport out of the ER, which drives
Assembly of the COPII coat is initiated by GDP-GTP exchange
on Sar1p catalyzed by the ER-localized transmembrane guanine
nucleotide exchange factor (GEF) Sec12p . GTP binding
induces a conformational change in Sar1p, which then inserts into
the ER membrane , . Membrane-bound Sar1p-GTP recruits
the Sec23/24p complex by binding to the Sec23p portion, and
Sec24p captures the cytoplasmically exposed ER export signal of
the transmembrane cargo ,  to form a prebudding complex
. The Sec23p subunit is the GTPase-activating protein (GAP)
for Sar1p , and therefore stimulates Sar1p GTP hydrolysis
upon binding to Sar1p, leading to disassembly of the prebudding
complex . However, even in the presence of ongoing GTP
hydrolysis, binding of Sec23/24p to membranes is stabilized
through interactions with transmembrane cargo proteins and
continual Sec12p-dependent GTP loading on Sar1p , .
Subsequently, the outer layer of the COPII coat consisting of
Sec13/31p is recruited onto the prebudding complex, which cross-
links adjacent prebudding complexes to drive membrane defor-
mation , and eventually, COPII vesicles are produced at the
specific subdomains of the ER known as ER exit sites (ERES) ,
, . The rate of GTP hydrolysis by Sar1p is further
accelerated through the binding of Sec13/31p to the prebudding
complex, which enhances the Sec23p-mediated GAP activity by
an order of magnitude. This activity has been shown to trigger
rapid disassembly of the fully assembled coat in a minimal system
. Additional factors are thought to regulate the Sar1p-GTPase
activity to prevent premature coat disassembly.
While we now know much about the molecular mechanisms of
COPII coat assembly and cargo selection, the physiological and
mechanistic features of regulation of ER-to-Golgi transport is a
relatively unexplored area. Since organelle compartment size and
number have been shown to reflect physiological changes,
including cell growth, differentiation, and response to stress ,
, , such compartment homeostasis must be flexible enough
PLoS ONE | www.plosone.org1 July 2012 | Volume 7 | Issue 7 | e40765
36. Wahl M, Sleight RG, Gruenstein E (1992) Association of cytoplasmic free Ca2+
gradients with subcellular organelles. J Cell Physiol 150: 593–609.
37. Camello C, Lomax R, Petersen OH, Tepikin AV (2002) Calcium leak from
intracellular stores–the enigma of calcium signalling. Cell Calcium 32: 355–361.
38. Pezzati R, Bossi M, Podini P, Meldolesi J, Grohovaz F (1997) High-resolution
calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane
system. Electron energy loss imaging analysis of quick frozen-freeze dried
PC12 cells. Mol Biol Cell 8: 1501–1512.
39. Cai H, Yu S, Menon S, Cai Y, Lazarova D, et al. (2007) TRAPPI tethers COPII
vesicles by binding the coat subunit Sec23. Nature 445: 941–944.
40. Matsuoka K, Orci L, Amherdt M, Bednarek SY, Hamamoto S, et al. (1998)
COPII-coated vesicle formation reconstituted with purified coat proteins and
chemically defined liposomes. Cell 93: 263–275.
41. Sato K, Nakano A (2004) Reconstitution of coat protein complex II (COPII)
vesicle formation from cargo-reconstituted proteoliposomes reveals the potential
role of GTP hydrolysis by Sar1p in protein sorting. J Biol Chem 279: 1330–
42. Kodera C, Yorimitsu T, Nakano A, Sato K (2011) Sed4p stimulates Sar1p GTP
hydrolysis and promotes limited coat disassembly. Traffic 12: 591–599.
Pef1p Regulates COPII Assembly
PLoS ONE | www.plosone.org8 July 2012 | Volume 7 | Issue 7 | e40765