Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

Alb, James G.; Phillips, Scott E.; Rostand, K; Cui, Xiaoxia; Pinxteren, Jef; Cotlin, Laura F.; Manning, Timothy J.; Guo, Shuling; York, John D.; Sontheimer, Harald; Collawn, James F.; Bankaitis, Vytas A.

doi:10.1091/mbc.01-09-0457

Cited by 65 publications

(71 citation statements)

References 61 publications

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“…To analyze PIPs in mammalian cells, control and hSAC1 small interfering RNA (siRNA)-treated HeLa cells were cultured in six-well plates, and radiolabeled to steady state with 100 Ci/ml [ 3 H]inositol (Ins) for 48 h. Cells were scraped, phospholipids (PLs) were extracted and deacylated with methylamine, and soluble glycerophosphoinositol species were resolved and quantified as described previously Rivas et al, 1999;Nemoto et al, 2000;Alb et al, 2002).…”

Section: Inositol Radiolabeling and Pip Analysesmentioning

confidence: 99%

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

Liu

Boukhelifa

Tribble

et al. 2008

MBoC

Self Cite

131

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Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties. INTRODUCTIONSpatial and temporal regulation of intracellular signaling in eukaryotic cells involves the compartmentalization of membrane surfaces into discrete, albeit often transient, functional units. There are several biochemical strategies by which cells generate such units or domains. One well-established strategy uses the chemical diversity offered by phosphoinositides (PIPs), i.e., phosphorylated forms of phosphatidylinositol (PtdIns) (Majerus, 1997;Fruman et al., 1998;Strahl and Thorner, 2007). The chemical heterogeneity of individual PIP species permits the construction of chemically unique platforms on membrane surfaces that, in turn, recruit unique cohorts of proteins that drive specific biological reactions. Spatial and temporal control of such reactions requires a finely coordinated balance between the activities of the lipid kinases that produce PIPs and the activities of enzymes that degrade them. PIP turnover is catalyzed by two general classes of enzymes: phospholipases and PIP phosphatases.Although experimental scrutiny of the phospholipases has historically been more intense, recent demonstrations of the significant biological functions played by PTEN, the myotubularins, and synaptojanins highlight the general importance of PIP phosphatases (Wishart et al., 2001;Wishart and Dixon, 2002;Wenk and De Camilli, 2004).The SAC domain derives from the yeast Sac1 protein (ySac1; Cleves et al., 1989), and it represents a signature for PIP phosphatase catalytic activity . PIP phosphatases such as phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) (Maehama et al., 2001), synaptojanins (Cremona et al., 1999), and synaptojanin-like proteins (Srinivasan et al., 1997;Stolz et al., 1998) all harbor SAC domains. The prototypical member of this family, ySac1, catalyzes the dephosphoryla...

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Section: Inositol Radiolabeling and Pip Analysesmentioning

confidence: 99%

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

Liu

Boukhelifa

Tribble

et al. 2008

MBoC

Self Cite

131

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“…In yeast, membrane flux through the exocytic pathway is regulated by cytosolic Sec14 [28], a family of cytosolic phosphatidylcholine/phosphatidylinositol transfer proteins that via their dual specificity may act as sensors of lipid composition and adapt lipid metabolism. Whereas one mammalian phosphatidylinositol transfer protein, PITPa, may have a related function and reports back to the nucleus [29], the highly homologous Golgi-associated PITPb is essential for growth [30]. PITPb binds sphingomyelin, which is unexpected as this lipid is thought to be confined to the Golgi lumen.…”

Section: Lipid-binding Proteins and Vesicle Fluxmentioning

confidence: 99%

“…Based on the prediction that the active center would be similar to that of the lipid phosphate phosphatase (LPP) family, the authors employ a functional cloning strategy in yeast to identify two mammalian sphingomyelin synthases, SMS1 in the Golgi and SMS2 at the plasma membrane, where it is presumably active in ceramide/diacylglycerol-mediated signaling events. [28][29][30]. This paper unravels the mechanism by which the ceramide transfer protein CERT specifically transports ceramide from the ER to the site of Golgi sphingomyelin synthesis [16 ].…”

Section: Updatementioning

confidence: 99%

Membrane lipids and vesicular traffic

Meer

Sprong

2004

Current Opinion in Cell Biology

157

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Lipids were long considered to be passive passengers of carrier vesicles with the single role of sealing the transport container. We now know that specific phospholipids are required for efficient fusion, while others facilitate budding and fission. Moreover, the various polyphosphoinositides assist in the recruitment from the cytosol of proteins of the transport machinery. Finally, the segregation of membrane lipids into different fluid phases appears to serve as a 'lipid raft' mechanism for protein sorting at various stages of the secretory and endocytic pathways. The current challenge is to understand how proteins control the metabolism and subcellular localization, and thereby the activity, of the various lipids.

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“…It seems that the decrease of PI-TP protein level may be one of the important factors that lead to disturbing of phospholipids signaling and also may be involved in neurodegeneration of CNS. Alb et al (2002Alb et al ( , 2003 indicated that lack of PI-TPα caused aponecrotic spinocerebrallar disease in PI-TPα knockout mice. The mice lacking PI-TPα die soon after birth (Alb et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylinositol Transfer Protein Expression Altered by Aging and Parkinson Disease

Chalimoniuk

Snoek²,

Adamczyk

et al. 2006

Cell Mol Neurobiol

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SUMMARY1. Phosphatidylinositol transfer proteins (PI-TP) are responsible for the transport of phosphatidylinositol (PI) and other phospholipids from endoplasmic reticulum to the other membranes and indirectly for lipid mediated signaling. Till now little is known about PITPs in brain aging and neurodegeneration. The aim of this study was to investigate expression of PI-TP in the brain during aging and in animal's model of Parkinson disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Moreover, in vitro, effect of 1-methyl-4-phenyl-pyridine cation (MPP + ) on PI-TP, tyrosine hydroxylase (TH) protein level, and viability of cells was investigated.2. Wistar rats 4, 24, and 36 months old and C57/BL mice and rat pheochromocytoma (PC12) cell line were used for the studies. Mice C57/BL received three injections of MPTP in saline at 2 h intervals in a total dose of 40 mg/kg and then after 3, 7, and 14 days they were used for the investigation. PC12 cells were treated with increasing concentration (50-300 µM) of MPP + for 24 h at 37 • C. The level of PI-TP α and β and TH were determined using Western Blot analysis.3. Our data indicated that PI-TP α and β level decreased in brain of 36 months old rat by 20% comparing to the control value (4 months old). In animal's model of PD, PI-TP α and β level was significantly lower by 85, 69, 64% in striatum at 3, 7, and 14 days after MPTP injection, respectively, compared to the control value. MPP + decreased PI-TP α and β , TH expression, and viability of PC12 cells in a dose-dependent manner. H 2 O 2 , menadione, and NO donor significantly decreased the PI-TP level and viability of PC12 cells.4. Our results indicate the lower protein expression of PI-TP α and β in aged brain and in PD and suggest that oxidative stress may be responsible for the alteration of PI-TP.

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Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

Cited by 65 publications

References 61 publications

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

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

Membrane lipids and vesicular traffic

Phosphatidylinositol Transfer Protein Expression Altered by Aging and Parkinson Disease

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