Direct Involvement of Phosphatidylinositol 4-Phosphate in Secretion in the Yeast Saccharomyces cerevisiae

Abstract

The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast. Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion. One suppressor, sac1-22, causes accumulation of PtdIns(4)P. SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides. These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect. Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37°C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34°C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases. This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells. To further address Pik1p function in secretion, two different pik1 ts mutants were examined. Upon shift to restrictive temperature (37°C), the PtdIns(4)P levels dropped by about 60% in both pik1 ts strains within 1 h. During the same period, cells displayed a reduction (40 -50%) in release of a secreted enzyme (invertase). However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y). These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-toplasma membrane stage of secretion.In eukaryotic cells, secreted proteins are synthesized on ribosomes targeted to the endoplasmic reticulum (ER), 1 translocated into the ER lumen, and transported through the secretory pathway (1). From the ER, secretory proteins are transported to the Golgi apparatus, through the subcompartments of the Golgi, and then to the cell surface or to certain intracellular organelles, all via small membrane-bound vesicles (transport vesicles) (2-4). Cargo proteins are packaged into transport vesicles that bud from one compartment and fuse with another. Mechanisms of vesicle budding and fusion are conserved from yeast to mammalian cells (3,5). Because of its tractability for genetic analysis, bakers' yeast (Saccharomyces cerevisiae) has proven to be a useful organism to identify gene products required for various events in secretion. Genetic screens, first applied by Schekman and co-workers (6), resulted in the isolation of temperature-sensitive sec mutants that displayed defects in different stages of secretion at the nonpermissive temperature. Characterization of the corresponding normal (SEC) genes has pinpointed many proteins necessary for secretory processes; and, a large number of gene products are now known to function at various steps in the secretory pathway (reviewed in Ref. 7). The SEC14 gene encodes a phosph...

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).…”

“…Substantial evidence demonstrates that both sec14 and pik1 (Hama et al, 1999;Walch-Solimena and Novick, 1999) mutations block vesicle-mediated transport from the Golgi. That both sec14-1 and pik1-ts diploids are unable to carry out meiosis suggests that trafficking through the secretory pathway is required for meiotic progression.…”

“…A second PtdIns 4-kinase, Stt4p (Yoshida et al, 1994), generates a distinct and essential pool of PtdIns(4)P (Audhya et al, 2000), whereas a third PtdIns 4-kinase (Lsb6p) has no essential function and is not required for sporulation (Han et al, 2002). There is substantial evidence that Sec14p contributes significantly to the pool of PtdIns that is converted to PtdIns(4)P in the Golgi via the action of Pik1p (Hama et al, 1999;Rivas et al, 1999).…”

“…Exit of secretory proteins from the Golgi requires the function of Sec14p, a PtdIns/PtdCho transfer protein (Bankaitis et al, , 1990, and Pik1p, a PtdIns 4-kinase, which phosphorylates PtdIns to synthesize PtdIns(4)P (Hama et al, 1999;Walch-Solimena and Novick, 1999;Audhya et al, 2000). Consequently, SEC14 and PIK1 are essential genes in vegetatively growing cells.…”

The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

“…mSac1-GFP showed a higher degree of colocalization with the cis-Golgi marker GM130 than with the TGN marker TGN46 ( Figure 3A). The presence of mSac1 in Golgi cisternae (particularly TGN) is paradoxical given PtdIns-4-P is important for anterograde trafficking from the TGN (Hama et al, 1999;Walch-Solimena and Novick, 1999;Godi et al, 2004). One possibility is mSac1 is disqualified from efficient entry into Golgi subregions from which anterograde vesicles bud.…”