Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24 -65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol-but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi‐derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP‐choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP‐choline pathway‐mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand‐binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi‐derived transport vesicle biogenesis.

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

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