VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus

Wyles, Jessica P.; Ridgway, Neale D.

doi:10.1016/j.yexcr.2004.03.052

Cited by 96 publications

(89 citation statements)

References 43 publications

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“…The affinity of binding by GST-VAP 1-142 was similar to that of full-length VAP just missing the transmembrane domain (13 and 11 M, respectively). These results indicate that the MSP-VAP domain is solely responsible for binding FFAT motifs, in agreement with a recent study (3).…”

Section: The Scs2 Homologue Scs22 Plays a Minor Role In Regulation Ofsupporting

confidence: 93%

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A Highly Conserved Binding Site in Vesicle-associated Membrane Protein-associated Protein (VAP) for the FFAT Motif of Lipid-binding Proteins

Loewen

Levine

2005

Journal of Biological Chemistry

217

220

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A variety of lipid-binding proteins contain a recently described motif, designated FFAT (two phenylalanines in an acidic tract), which binds to vesicle-associatedmembrane protein-associated protein (VAP). VAP is a conserved integral membrane protein of the endoplasmic reticulum that contains at its amino terminus a domain related to the major sperm protein of nematode worms. Here we have studied the FFAT-VAP interaction in Saccharomyces cerevisiae, where the VAP homologue Scs2 regulates phospholipid metabolism via an interaction with the FFAT motif of Opi1. By introducing mutations at random into Scs2, we found that mutations that abrogated binding to FFAT were clustered in the most highly conserved region. Using site-directed mutagenesis, we identified several critical residues, including two lysines widely separated in the primary sequence. By examining all other conserved basic residues, we identified a third residue that was moderately important for binding FFAT. Modeling VAP on the known structure of major sperm protein showed that the critical residues form a patch on a positively charged face of the protein. In vivo functional studies of SCS22, a second SCS2-like gene in S. cerevisiae, showed that SCS2 was the dominant gene in the regulation of Opi1, with a minor contribution from SCS22. We then established that reduction in the affinity of Scs2 mutants for FFAT correlated well with loss of function, indicating the importance of these residues for binding FFAT motifs. Finally, we found that human VAP-A could substitute for Scs2 but that it functioned poorly, suggesting that other factors modulate the binding of Scs2 to proteins with FFAT motifs.

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Section: The Scs2 Homologue Scs22 Plays a Minor Role In Regulation Ofsupporting

confidence: 93%

“…they contain domains that stabilize hydrophobic lipids in the aqueous environment of the cytoplasm and thus can carry lipid traffic across the cytoplasm (2). Most of these have now been shown to bind VAP (3,4). Therefore, a large number of lipid traffic steps into/out of the ER are potentially mediated by FFAT-VAP interactions.…”

mentioning

confidence: 99%

A Highly Conserved Binding Site in Vesicle-associated Membrane Protein-associated Protein (VAP) for the FFAT Motif of Lipid-binding Proteins

Loewen

Levine

2005

Journal of Biological Chemistry

217

220

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“…For some of them, physical interactions with VAPs have been demonstrated (9,15,20,21). We here showed for the first time that the FFAT motif of the ceramide transfer protein CERT is actually important for the ER-to-Golgi trafficking activity of CERT.…”

Section: Discussionmentioning

confidence: 61%

Efficient Trafficking of Ceramide from the Endoplasmic Reticulum to the Golgi Apparatus Requires a VAMP-associated Protein-interacting FFAT Motif of CERT

Kawano

Kumagai

Nishijima

et al. 2006

Journal of Biological Chemistry

279

301

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Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleckstrin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ERresident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.The transport and sorting of lipids from the sites of their synthesis to their appropriate destinations are essential events for membrane biogenesis in cells. In the synthesis of sphingolipids in mammalian cells, ceramide is newly produced at the endoplasmic reticulum (ER) 2 and transported from the ER to the trans-Golgi regions, where it is converted to sphingomyelin (SM) by the enzyme phosphatidylcholine:ceramide cholinephosphotransferase (SM synthase). Ceramide is also converted to glucosylceramide (GlcCer) presumably in the cis-Golgi regions or a subdomain of the ER. GlcCer is converted to more complex glycosphingolipids in the Golgi apparatus. The major transport of ceramide from the ER to the Golgi apparatus for the synthesis of SM is an ATP-and cytosol-dependent pathway (1, 2) and is genetically impaired in a Chinese hamster ovary (CHO) mutant cell line named LY-A (1, 2). After functional gene rescue experiments, the factor mutated in LY-A cells was identified to be CERT (also known as Goodpasture antigen-binding protein), a 68-kDa cytosolic protein (3). CERT consists of three distinct regions. The N-terminal ϳ120 residues of CERT form a pleckstrin homology (PH) domain, which is a phosphoinositide-binding domain (4, 5). The C-terminal ϳ230 residues form a START domain, a putative lipid transfer domain (6). The middle region, the ϳ250 amino acid residues between the PH and START domains, is predicted to form no globular domains.The PH domain of CERT serves to target the Golgi apparatus by recognizing phosphatidylinositol 4-monophosphate (PI4P) (3, 7). The START domain of CERT is capable of extracting ceramide from membranes and transferring it to acceptor membranes (3,8). Based on these findings, we have proposed that CERT extracts ceramide fr...

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“…Association of endogenous FAPP1 and FAPP2 with HeLa TGN was similarly undisturbed (data not shown). Localization of Orp9, a Golgi-targeted member of the oxysterol binding protein family (Lessmann et al, 2007;Wyles and Ridgway, 2004), was also unimpressed by hSac1 depletion ( Figure 7B). The structurally deranged Golgi system in hSac1-depleted cells retained its cohort of other peripheral Golgi proteins as well, including ␤-COP (Chen et al, 2005;Duden et al, 1991;Oprins et al, 1993) (Figure 7C) and the TGN-associated phosphatidylinositol transfer protein (PITP)␤ (Phillips et al, 2006) (Supplemental Figure S4C).…”

Section: Disorganized Golgi Retain Transport Competencementioning

confidence: 99%

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

Liu

Boukhelifa

Tribble

et al. 2008

MBoC

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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VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus

Cited by 96 publications

References 43 publications

A Highly Conserved Binding Site in Vesicle-associated Membrane Protein-associated Protein (VAP) for the FFAT Motif of Lipid-binding Proteins

A Highly Conserved Binding Site in Vesicle-associated Membrane Protein-associated Protein (VAP) for the FFAT Motif of Lipid-binding Proteins

Efficient Trafficking of Ceramide from the Endoplasmic Reticulum to the Golgi Apparatus Requires a VAMP-associated Protein-interacting FFAT Motif of CERT

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

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