SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.

Yeast phosphatidylinositol transfer protein (Sec14p) is required for the production of secretory vesicles from the Golgi. This requirement can be relieved by inactivation of the cytosine 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis, indicating that Sec14p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Sec14p pathway). Sac1p (refs 7 and 8) is an integral membrane protein related to inositol-5-phosphatases such as synaptojanin, a protein found in rat brain. Here we show that defects in Sac1p also relieve the requirement for Sec14p by altering phospholipid metabolism so as to expand the pool of diacylglycerol (DAG) in the Golgi. Moreover, although short-chain DAG improves secretory function in strains with a temperature-sensitive Sec14p, expression of diacylglycerol kinase from Escherichia coli further impairs it. The essential function of Sec14p may therefore be to maintain a sufficient pool of DAG in the Golgi to support the production of secretory vesicles.

Abstract. Mutations in the SAC1 gene exhibit allelespecific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAClp is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sad mutants result from loss of SAClp function. This cosuppression of actin and SEC14p defects is a unique feature of sad alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sad-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sad mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAClp dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids.

Abstract. SEC14p is required for protein transport from the yeast Golgi complex. We describe a quantitative analysis of yeast bulk membrane and Golgi membrane phospholipid composition under conditions where Golgi secretory function has been uncoupled from its usual SEC14p requirement. The data demonstrate that SEC14p specifically functions to maintain a reduced phosphatidylcholine content in Golgi membranes and indicate that overproduction of SEC14p markedly reduces the apparent rate of phosphatidylcholine biosynthesis via the CDP-choline pathway in vivo. We suggest that SEC14p serves as a sensor of Golgi membrane phospholipid composition through which the activity of the CDP-choline pathway in Golgi membranes is regulated such that a phosphatidylcholine content that is compatible with the essential secretory function of these membranes is maintained.T HE Saccharomyces cerevisiae SEC14 gene product (SEC14p) is a phosphatidylinositol/phosphatidylcholine transfer protein whose function is essential for both protein transport from a late yeast Golgi compartment and for yeast viability (Bankaitis et al., , 1990Franzusoff and Schekman, 1989;Aitken et al., 1990). As such, SEC14p has provided a system with which to study the in vivo function of a ubiquitous class of enigmatic proteins, the phospholipid transfer proteins, that are operationally defined by their ability to serve as diffusible carders of phospholipid monomers between membrane bilayers in vitro (reviewed by Rueckert and Schmidt, 1990;Wirtz, 1991; Cleves et ai., 1991a). We consider SEC14p to play a direct role in yeast Golgi secretory function as this protein is found in both a cytoplasmic pool and in a stable, and apparently specific, peripheral association with the yeast Golgi complex Cleves et al., 1991b).To date, the most instructive clues relating to SECI4p function in vivo have been obtained from analyzes of yeast mutants that no longer require SEC14p in order to survive and efficiently execute Golgi secretory function (Cleves et al., 1989(Cleves et al., , 1991b. These studies revealed that one unanticipated mechanism for bypass of SEC14p function involves inactivation of the cytosine-diphosphate (CDP)-choline pathway for phosphatidylcholine (PC) ~ biosynthesis, a pathway that consists of three reactions resulting in the incorporation of choline into PC (Kennedy and Weiss, 1956; see Fig. 1 A). The finding that the cellular requirement for SEC14p is obviated by inactivation of a specific avenue for PC biosynthesis demonstrated, for the first time, a direct physiological relationship between an in vitro ligand of a phospholipid transfer protein and the function of that phospholipid transfer protein in vivo. The collective SEC14p data, including the data indicating that SEC14p prefers phosphatidylinositol (PI) over PC as a substrate in the in vitro transfer reaction (Daurn and Paltauf, 1984), have been reconciled in the PI/ PC hypothesis for SEC14p function in vivo (Cleves et al., 1991a,b). The PI/PC hypothesis proposes that SEC14p functions to maintai...

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