Phospholipid transfer activity is relevant to but not sufficient for the essential function of the yeast SEC14 gene product.

Skinner, Henry B.; Alb, James G.; Whitters, Eric A.; Helmkamp, George M.; Bankaitis, Vytas A.

doi:10.1002/j.1460-2075.1993.tb06166.x

Cited by 112 publications

(70 citation statements)

References 23 publications

(38 reference statements)

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“…The activity of the PITP␤-GFP chimera was established with a yeast phenotypic rescue assay. This assay capitalizes on previous demonstrations that high-level expression of mammalian PITPs in yeast rescues the growth and secretory defects associated with inactivation of the essential yeast PITP Sec14p (Skinner et al, 1993;Tanaka and Hosaka, 1994). This rescue is dependent on robust PtdInsbinding/transfer by the heterologous mammalian PITP (Alb et al, 1995).…”

Section: Endogenous Pitp␤ Localizes To the Mammalian Golgi Complexmentioning

confidence: 80%

“…The HindIII-BamHI PCR fragments were cloned into the pEGFP-C1 plasmid (Clontech, Palo Alto, CA). Yeast plasmids harboring PITP␣ and PITP␤ cDNAs (Skinner et al, 1993) were used as templates in the PCR reactions used for generating the appropriate DNA fragments for cloning. The resulting plasmids were designated pRE772 (PITP␤-GFP) and pRE774 (PITP␣-GFP).…”

Section: Generation Of Pitp␣-gfp and Pitp␤-gfp Cdnasmentioning

confidence: 99%

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Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the MammalianTrans-Golgi Network

Phillips

Ile

Boukhelifa

et al. 2006

MBoC

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Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITP␤ localization in mammalian cells. We demonstrate PITP␤ localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITP␤. Domain mapping analyses show the targeting information within PITP␤ consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W 202 W 203 motif). Combination of the specificity elements with the W 202 W 203 motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITP␤ association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITP␤ isoform with a naturally occurring S 262 Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITP␤ localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms ␦ or . These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser 262 is required for PITP␤ targeting to Golgi membranes.

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Section: Endogenous Pitp␤ Localizes To the Mammalian Golgi Complexmentioning

confidence: 80%

Section: Generation Of Pitp␣-gfp and Pitp␤-gfp Cdnasmentioning

confidence: 99%

Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the MammalianTrans-Golgi Network

Phillips

Ile

Boukhelifa

et al. 2006

MBoC

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“…This mechanism accounts for the enhancement of phosphoinositide levels at the plasma membrane by at least one mammalian PITP (51). A rat PtdIns/PtdCho exchange protein that bears little sequence similarity to Sec14p is nonetheless able to complement a sec14 temperature-sensitive mutant in vivo (52), suggesting that the phospholipid exchange activity of this heterologous PITP is responsible for the phenotypic rescue rather than its specific contacts with any yeast protein. In any event, whether Sec14p and Pik1p interact and are localized to the Golgi in yeast are important issues for future study.…”

Section: Delivery Of Cpy To the Vacuole Is Not Blocked In Pik1 Tsmentioning

confidence: 99%

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

Hama¹,

Schnieders²,

Thorner³

et al. 1999

Journal of Biological Chemistry

265

268

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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...

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“…SEC14p, although able to transfer PI and PC does not bear any sequence homology to the mammalian forms of PITP (2). However, temperature-sensitive secl4 mutants could be rescued by PITP,3 as well as PITPa (10,25).…”

mentioning

confidence: 97%

“…SEC14p and PITP,B are localized at the Golgi, suggesting that they may play a specific role in this compartment (11,(25)(26)(27) (9).…”

mentioning

confidence: 99%

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

Cunningham

Tan

Swigart

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

107

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The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPa is required to restore G protein-mediated phospholipase Cf8 (PLCI3) signaling. Recently, a second mammalian PITP13 form has been described that shows 77% identity to rat PITPa. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPa and PITPjJ isoforms as well as SEC14p restore G protein-mediated PLCI8 signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCyl. Permeabilization of RBL cells leads to loss of PLCyl as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLC'y1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

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Phospholipid transfer activity is relevant to but not sufficient for the essential function of the yeast SEC14 gene product.

Cited by 112 publications

References 23 publications

Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the MammalianTrans-Golgi Network

Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the MammalianTrans-Golgi Network

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

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

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