Mammalian PIG-X and Yeast Pbn1p Are the Essential Components of Glycosylphosphatidylinositol-Mannosyltransferase I

Ashida, Hisashi; Hong, Yeongjin; Murakami, Yoshiko; Shishioh, Nobue; Sugimoto, Nakaba; Kim, Youn Uck; Maeda, Yusuke; Kinoshita, Taroh

doi:10.1091/mbc.e04-09-0802

Cited by 67 publications

(61 citation statements)

References 34 publications

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“…Cells of the F21 background stably express the GPI anchor marker proteins CD59 and DAF and twelve proteins involved in GPI anchor biosynthesis ( 20 ) whereas cells of the C311 background express four proteins of the GPI anchor biosynthesis pathway in addition to the markers CD59 and DAF (21)(22)(23). Presence of those plasmids was verifi ed by antibiotic resistance of cells to G418, hygromycin B and blasticidin S (F21 series) or resistance of cells to G418, hygromycin B and puromycin (C311 series).…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Loizides‐Mangold

David

Nesatyy

et al. 2012

Journal of Lipid Research

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This article is available online at http://www.jlr.org anchor has the core structure phosphatidylinositol (PI)-glucosamine (GlcN)-(Mannose) 3 -phosphoethanolamine (EtN-P), which is conserved among all species. After biosynthesis, the GPI anchor is attached posttranslationally to the newly generated C terminus of certain eukaryotic proteins destined for anchoring thereby tethering the protein to the membrane surface by the glycolipid moiety. GPI-anchored proteins can be released from the cell surface by phosphatidylinositol specifi c phospholipases and this cleavage event can induce major conformational changes on the GPIanchored protein itself ( 2 ).At least three organelles, the endoplasmic reticulum (ER), Golgi, and peroxisomes, are involved in the biosynthesis and remodeling of the GPI anchor. The biosynthesis is initiated on the outer side of the ER membrane. After the fi rst two reactions, the GPI anchor precursor is fl ipped and biosynthesis continues on the luminal side of the ER where the diacyl chains of phosphatidylinositol are then replaced by alkyl-acyl chains. This step is impaired in mutants of the peroxisomal alkyl phospholipid biosynthesis pathway ( 3 ).After protein attachment, the GPI anchor undergoes complex remodeling that begins in the ER with the removal of the inositol-linked acyl chain ( 4 ) and the remodeling of the GPI glycan part (5). Glycan remodeling is crucial for sorting GPI-anchored proteins into ER exit sites and their subsequent ER to Golgi transport ( 6 ). In mammalian cells, remodeling of the GPI anchor is then continued in the Golgi where the unsaturated fatty acid of the GPI anchor is replaced by a saturated fatty acid chain ( 7 ). Abstract Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER).After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profi le of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchordefi cient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-defi cient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by...

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Section: Cell Culture and Transfectionmentioning

confidence: 99%

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Loizides‐Mangold

David

Nesatyy

et al. 2012

Journal of Lipid Research

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“…However, we do not know what the significance of the homology between Pbn1p and PIG-X is in relation to our results. The following points are worth noting: (i) the mammalian and yeast proteins are Ͻ20% identical in the overlapping region, (ii) complementation of the PIG-X mutant phenotype was not achieved by transfection with PBN1 alone but required cotransfection with another gene, GPI14, the catalytic component of GPI-MT I in yeast (8), (iii) in the absence of Pbn1p, the processing of a GPI-anchored protein Gas1p was affected, but the defect was in conversion of the ER to the Golgi form, and (iv) absence of Pbn1p abrogates or impairs the processing of precursors to at least two proteins in yeast that lack GPI anchors.…”

Section: Discussionmentioning

confidence: 99%

“…A recent report, published during the preparation of this manuscript, indicates that Pbn1p may be a functional homologue of the protein PIG-X in higher eukaryotes (8). PIG-X is a component of the glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT I) complex and is involved in the addition of the first mannose residue to the GPI anchor precursor.…”

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confidence: 99%

Pbn1p: An essential endoplasmic reticulum membrane protein required for protein processing in the endoplasmic reticulum of budding yeast

Subramanian¹,

Woolford²,

Drill³

et al. 2006

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

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PBN1 was identified as a gene required for production of protease B (PrB) activity in Saccharomyces cerevisiae. PBN1 encodes an endoplasmic reticulum (ER)-localized, type I membrane glycoprotein and is essential for cell viability. To study the essential function(s) of Pbn1p, we constructed a strain with PBN1 under control of the GAL promoter. Depletion of Pbn1p in this strain abrogates processing of the ER precursor forms of PrB, Gas1p, and Pho8p. Depletion of Pbn1p does not affect exit of proprotease A or procarboxypeptidase Y from the ER, indicating that Pbn1p is not required for global exit from the ER. Depleting Pbn1p leads to a significant increase in the unfolded protein response pathway, accompanied by an expansion of bulk ER membrane, indicating that there is a defect in protein folding in the ER. pbn1-1, a nonlethal allele of PBN1, displays synthetic lethality with the ero1-1 allele (ERO1 is required for oxidation in the ER) and synthetic growth defects with the cne1⌬ allele (CNE1 encodes calnexin). ER-associated degradation of a lumenal substrate, CPY*, is blocked in the absence of Pbn1p. These results suggest that Pbn1p is required for proper folding and͞or the stability of a subset of proteins in the ER. Thus, Pbn1p is an essential chaperone-like protein in the ER of yeast.chaperone ͉ protein folding ͉ unfolded protein response T he endoplasmic reticulum (ER) in eukaryotes is home to a variety of chaperones and enzymes that monitor the status of every protein that enters the secretory pathway (1). With the help of these chaperones and enzymes, transiting proteins and ER resident proteins are folded properly into their native conformations. This quality-control pathway also includes protein disulfide-bond formation and glycosylation. Once folded, the proteins are either retained in the ER or transported to the Golgi apparatus, from which they travel to their final destinations. Many factors can perturb this ER folding machinery, including inhibition of N-linked glycosylation, treatment with calcium ionophores, alteration in the oxidation state of the ER, and misfolding of secretory proteins, leading to a block in exit from the ER. In most cases, these kinds of perturbations induce the unfolded protein response (UPR) pathway; as a result, transcription of chaperone genes and other genes involved in protein folding are up-regulated (2, 3). In some cases, misfolding of proteins leads to retrotranslocation from the ER and subsequent degradation of the protein by the cytoplasmic proteasome via a pathway called ER-associated degradation (ERAD) (4).PBN1 is an essential gene that was identified in a screen for mutants that were deficient for protease B (PrB) activity (5). Pbn1p is a type I membrane glycoprotein and resides in the ER of yeast. No phenotypic change other than PrB deficiency and a mild sensitivity to the reducing agent DTT has been associated with the pbn1-1 allele (5). The PrB precursor undergoes glycosylation and four proteolytic cleavages during maturation (6, 7). The pbn1-1 strain lacks PrB...

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“…To test the possibility that Pga1 has genetic interaction with other GPI-MTs, we introduced GPI14, PBN1, GPI10, or SMP3 on 2 plasmid. GPI14 and PBN1 encode subunits of GPI-MT-I (Maeda et al, 2001;Ashida et al, 2005), GPI10 encodes the third ␣1,2-mannosyltransferase, and SMP3 encodes the last ␣1,2-mannosyltransferase (Grimme et al, 2001). However, multicopy expression of each gene could not restore the growth of the pga1-1 mutant at 37°C ( Figure 6B).…”

Section: Interaction Of Pga1 With Gpi18 An Essential Glycosylphosphamentioning

confidence: 99%

“…The enzymes that add Man to GPI precursors are called GPI-mannosyltransferase (GPI-MT) I, -II, -III, and -IV, according to the order of mannose addition. Each GPI-MT consists of at least one protein with several transmembrane domains that has homology to other enzymes responsible for glycosylation (Takahashi et al, 1996;Grimme et al, 2001;Maeda et al, 2001;Ashida et al, 2005;Fabre et al, 2005;Kang et al, 2005). So far, GPI-MT I has been the only enzyme that is found to consist of multisubunits.…”

Section: Introductionmentioning

confidence: 99%

Pga1 Is an Essential Component of Glycosylphosphatidylinositol-Mannosyltransferase II ofSaccharomyces cerevisiae

Sato

Noda

Yoda

2007

MBoC

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The Saccharomyces cerevisiae essential gene YNL158w/PGA1 encodes an endoplasmic reticulum (ER)-localized membrane protein. We constructed temperature-sensitive alleles of PGA1 by error-prone polymerase chain reaction mutagenesis to explore its biological role. Pulse-chase experiments revealed that the pga1 ts mutants accumulated the ER-form precursor of Gas1 protein at the restrictive temperature. Transport of invertase and carboxypeptidase Y were not affected. Triton X-114 phase separation and [ 3 H]inositol labeling indicated that the glycosylphosphatidylinositol (GPI)-anchoring was defective in the pga1 ts mutants, suggesting that Pga1 is involved in GPI synthesis or its transfer to target proteins. We found GPI18, which was recently reported to encode GPI-mannosyltransferase II (GPI-MT II), as a high-copy suppressor of the temperature sensitivity of pga1 ts . Both Gpi18 and Pga1 were detected in the ER by immunofluorescence, and they were coprecipitated from the Triton X-100 -solubilized membrane. The gpi18 ts and pga1 ts mutants accumulated the same GPI synthetic intermediate at the restrictive temperature. From these results, we concluded that Pga1 is an additional essential component of the yeast GPI-MT II.

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Mammalian PIG-X and Yeast Pbn1p Are the Essential Components of Glycosylphosphatidylinositol-Mannosyltransferase I

Cited by 67 publications

References 34 publications

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Pbn1p: An essential endoplasmic reticulum membrane protein required for protein processing in the endoplasmic reticulum of budding yeast

Pga1 Is an Essential Component of Glycosylphosphatidylinositol-Mannosyltransferase II ofSaccharomyces cerevisiae

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