Identification of a Novel Family of Nonclassic Yeast Phosphatidylinositol Transfer Proteins Whose Function Modulates Phospholipase D Activity and Sec14p-independent Cell Growth

Abstract

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.

Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation

“…Consistent with this suggestion, increased expression of Shf2p and Shf4p, yeast PITPs that display PtdIns-but not PtdCho-transfer activity in vitro, can rescue the growth and secretory defects of sec14 mutants and requires the function of Spo14p to do so (Li et al, 2000). However, Sec14p itself exhibits both PtdCho and PtdIns transfer activity in vitro (Bankaitis et al, 1990), and the results of genetic analyses have not supported a model in which the essential vegetative function of Sec14p is to provide for activation of Spo14p (Sreenivas et al, 1998;Xie et al, 1998), because unlike sec14⌬ mutants, spo14⌬ mutants are viable (Honigberg et al, 1992;Rose et al, 1995).…”

“…That the suppression by SFH2 or SFH4 is dependent on Spo14p indicates that Spo14p PLD activity can substitute for Sec14p under these conditions. This may be due to activation of Spo14p through enhanced PtdIns(4,5)P 2 generation, as observed in Sec14 bypass conditions (Li et al, 2000), and may be a consequence of Spo14p-dependent PtdCho cleavage mimicking Sec14p-PtdCho transport function.…”

“…In vegetative cells, two of these, Sfh2/Csr1p (Li et al, 2000;Santos and Snyder, 2000) and Sfh4/Pdr17p (van den Hazel et al, 1999;Li et al, 2000), potently suppress the sec14-1 lethality when overexpressed, and this suppression is dependent on Spo14p (Li et al, 2000). Likewise, these proteins suppressed the sec14-1 meiosis and spore-formation defects ( Table 2, lines 15 and 17), and the suppression of the meiotic defect was dependent on Spo14p function (Table 2, lines 16 and 18).…”

Nodule-Specific Regulation of Phosphatidylinositol Transfer Protein Expression in Lotus japonicus

“…Li et al, 2000), we further investigated the relationship between the Sec14 domain of LjPLP-IV and the yeast Sec14 protein on a functional level.…”

“…Studies in a number of eukaryotic systems have demonstrated that PITPs function to regulate various aspects of lipid metabolism (reviewed in Cleves et al, 1991; B.G. Li et al, 2000), but the first clue about their significance in vivo emerged from studies of Saccharomyces cerevisiae (Novick et al, 1980;Bankaitis et al, 1989Bankaitis et al, , 1990Aitken et al, 1990) showing their essential role in the formation of secretory vesicles and in protein transport from the Golgi complex. In vitro studies in mammalian systems have suggested that PITPs play important roles in promoting the activities of various inositol lipid-signaling pathways by regulating the production of certain phosphoinositides (Hay and Martin, 1993;Hay et al, 1995;Ohashi et al, 1995;Cunningham et al, 1996;Jones et al, 1998;Simon et al, 1998).…”