Ceramide biosynthesis is required for the formation of the oligomeric H+-ATPase Pma1p in the yeast endoplasmic reticulum.
The yeast plasma membrane H(+)-ATPase Pma1p is one of the most abundant proteins to traverse the secretory pathway. Newly synthesized Pma1p exits the endoplasmic reticulum (ER) via COPII-coated vesicles bound for the Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a unique role for Lst1p in ER export. Vesicles formed with mixed Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we examined the relationship between Pma1p biosynthesis and the requirement for this novel coat subunit. We show that Pma1p forms a large oligomeric complex of >1 MDa in the ER, which is packaged into COPII vesicles. Furthermore, oligomerization of Pma1p is linked to membrane lipid composition; Pma1p is rendered monomeric in cells depleted of ceramide, suggesting that association with lipid rafts may influence oligomerization. Surprisingly, monomeric Pma1p present in ceramide-deficient membranes can be exported from the ER in COPII vesicles in a reaction that is stimulated by Lst1p. We suggest that Lst1p directly conveys Pma1p into a COPII vesicle and that the larger size of mixed Sec24pLst1p COPII vesicles is not essential to the packaging of large oligomeric complexes.
Lee
Marcus C S
MC
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
Hamamoto
Susan
S
Schekman
Randy
R
D022181:COP-Coated Vesicles / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000096:biosynthesis* / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D050356:Lipid Metabolism; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D015003:Yeasts / Q000378:metabolism
D002518:Ceramides; D003902:Detergents; D029701:Saccharomyces cerevisiae Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases