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32
Sec31p as a sole component of the COPII cage:
understanding the bst phenotype
Catherine Latham 1 , Alenka Copic 1 , Mariella Ravazolla 2 , Lelio Orci 2 , Elizabeth Miller 1 .
Department of Biological Science, Columbia University, New York, NY, 10027 1 , Department of
Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland 2
Abstract:
Nascent proteins that enter the secretory pathway via the endoplasmic reticulum (ER) are
packaged into vesicles for forward transport. Vesicles are sculpted from the ER membrane by
the cytosolic COPII protein coat - the G-protein, Sar1p, Sec23/Sec24p and Sec13/Sec31p - in a
process that is highly conserved among eukaryotes. In budding yeast, these COPII proteins are
essential for viability. However, in certain mutants, called bypass of Sec thirteen (bst), cells can
survive without SEC13 (1). All known bst mutations affect biogenesis or trafficking of GPIanchored
proteins. We are currently exploring the mechanistic basis of the bst phenotype. One
hypothesis is that Sec31p can form the outer coat without Sec13p. In support of this model,
structural studies suggest that Sec31p forms many of the critical contacts of the outer coat,
including the intermolecular contacts of the Sec13/31p cage and interactions with the inner coat
proteins (2,3). We have purified monomeric Sec31p from an insect cell expression system and
demonstrate that Sec31p is specifically recruited by Sar1p and Sec23/24p to synthetic liposomes
in the absence of Sec13p. Monomeric Sec13p is not recruited to liposomes under similar
experimental conditions, whereas a Sec31p mutant that does not bind to Sec13p retains the
ability to form a coat complex with Sar1/Sec23/Sec24p. We are currently investigating the
structural morphology of vesicles produced by this Sec13p-deficient COPII coat, as well as the
functionality of Sec31p in an in vitro budding assay without Sec13p. Defining the mechanistic
basis of the bst phenotype contributes to our understanding of the link between the membrane
environment and COPII coat function. 1.Elrod-Erickson et al, Mol Biol Cell(1996)7:1043-58.
2.Bi et al. Dev Cell(2007)13:635 3.Stagg et al. Cell(2008)134:474.