A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes

Abstract

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...

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

“…However, we have found that PS-deficient yeast (cho1) produces these vesicles normally and transports proteins by means of the secretory pathway with WT kinetics. This PS-independence of protein transport is remarkable, considering that PS comprises Ϸ20% of the yeast Golgi complex membrane (33). Moreover, Drs2p is still required for the formation of exocytic vesicles in the absence of PS, indicating that compensatory changes in cho1 phospholipid composition do not bypass the requirement for Drs2p in protein transport.…”

Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects

“…PLD activity might support 'bypass Sec14p' because it reduces PtdCho levels in Golgi membranes (3). Alternatively, PLD might generate a lipid precursor that is limiting in the Golgi membranes of Sec14p-deficient cells (4,7,9). We wished to address which effect defined the relevant contribution of PLD to 'bypass Sec14p'.…”

“…The case for how Sec14p stimulates Golgi secretory function is derived primarily from analyses of mutations that permit efficient Golgi secretory activity (and cell viability) in the face of sec14 deletion mutations (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Characterization of these 'bypass Sec14p' mutations suggests that Sec14p maintains the integrity of a critical Golgi diacylglycerol (DAG) pool that is somehow required for Golgi secretory function (4,7).…”

“…First, 'bypass Sec14p' is generated by genetic inactivation of the cytidine 5Ј-diphosphate (CDP)-choline pathway for phosphatidylcholine (PtdCho) biosynthesis; a pathway that consumes DAG (3). Biochemical insight into this genetic relationship was provided by the demonstration that Sec14p is an inhibitor of the CDP-choline pathway (4,5). Second, accelerated PtdInsmetabolism represents another in vivo mechanism for effecting a 'bypass Sec14p' phenotype (7,9).…”

Phosphatidylinositol transfer proteins: From closed for transport to open for exchange

“…At this point it is still questionable whether these proteins actually transfer these phospholipids between membranes within cells or whether they interact directly with enzymes like PI 4-kinases and PI-specific phospholipase A 2 thereby providing these enzymes with substrates Snoek et al, 1999). In analogy with observations in yeast it has also been proposed that by binding either PI, PC or SPM, PI-TPs act as regulators of phospholipid metabolism at distinct sites in the cell thereby fulfilling the roles of biosensors (McGee et al, 1994;Bankaitis, 2002). Although much remains to be elucidated about their physiological role an observation to be accounted for is that PI-TPa localizes predominantly to the nucleus and cytoplasm whilst PI-TPb is mainly associated with the Golgi complex (De Vries et al, 1995, 1996.…”