Yeast Sec14p Deficient in Phosphatidylinositol Transfer Activity Is Functional In Vivo

Abstract

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

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

“…In vegetative sec14(K66A K239A) cells, the synthesis of PtdIns(4)P is reduced (Phillips et al, 1999), and overexpression of the PtdIns 4-kinase Pik1p suppresses the growth defect of sec14-1 cells at semirestrictive temperature (Hama et al, 1999). Similarly, we found that overexpression of PIK1 could restore the sporulation of sec14-1 diploids to near wild-type frequency, and suppression of the meiotic defect was dependent on Spo14p (Table 2, lines 19 -20).…”

“…Thus, the reduction in Spo14p activity observed in sec14-1 cells could be due to the loss of Sec14p-dependent transport of the substrate, PtdCho, to the cellular compartment(s) where Spo14p functions, to loss of Sec14p-dependent transport of PtdIns and the consequent failure to synthesize the PLD activator PtdIns(4,5)P 2 , or both. To explore the relative contributions of PtdIns and PtdCho binding/transport by Sec14p, we took advantage of a mutant, sec14(K66A K239A), that retains the ability to bind and transport PtdCho but is specifically defective in PtdIns binding and transport (Phillips et al, 1999). This mutant supports the essential function of Sec14p for growth and secretion in vegetative cells (Phillips et al, 1999).…”

“…Site-directed mutagenesis was performed to generate the double mutant sec14(K66A K239A) (pME1946), using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) as recommended by the manufacturer. The mutagenic primers were identical to those described by Phillips et al (1999). SEC14 and sec14(K66A K239A) were each subcloned into a multicopy (2 DNA-based) yeast vector, YEp352 (Hill et al, 1986), as ϳ3.5-kb XbaI-HindIII fragments to generate pME1881 and pME2062, respectively.…”

“…To explore the relative contributions of PtdIns and PtdCho binding/transport by Sec14p, we took advantage of a mutant, sec14(K66A K239A), that retains the ability to bind and transport PtdCho but is specifically defective in PtdIns binding and transport (Phillips et al, 1999). This mutant supports the essential function of Sec14p for growth and secretion in vegetative cells (Phillips et al, 1999). However, when expressed in sec14-1 cells at near-normal levels from a lowcopy-number plasmid, this mutant supported only very low levels of spore formation at the nonpermissive temperature (Table 2, line 13).…”

Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae Sec14p are found in the same cell

“…Analysis of bypass mutants has led to the suggestion that Sec14p is required for maintenance of a critical pool of diacylglycerol [21,22]. Moreover, Sec14p can also fa-cilitate PtdIns4P production in i o [22,23] ; this lipid might also be a critical requirement in protein secretion. In another yeast, Yarrowia lipolytica, SEC14 is not an essential gene and is required only for differentiation from yeast to a mycelial growth form [24].…”