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Cdc1p is a Golgi-localized glycosylphosphatidylinositol-anchored protein remodelase.

ABSTRACT: Glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive posttranslational modifications and remodeling, including the addition and subsequent removal of phosphoethanolamine (EtNP) from mannose 1 (Man1) and mannose 2 (Man2) of the glycan moiety. Removal of EtNP from Man1 is catalyzed by Cdc1p, an event that has previously been considered to occur in the endoplasmic reticulum (ER). We establish that Cdc1p is in fact a cis/medial Golgi membrane protein that relies on the COPI coatomer for its retention in this organelle. We also determine that Cdc1p does not cycle between the Golgi and the ER, and consistent with this finding, when expressed at endogenous levels ER-localized Cdc1p-HDEL is unable to support the growth of cdc1Δ cells. Our cdc1 temperature-sensitive alleles are defective in the transport of a prototypical GPI-AP-Gas1p to the cell surface, a finding we posit reveals a novel Golgi-localized quality control warrant. Thus, yeast cells scrutinize GPI-APs in the ER and also in the Golgi, where removal of EtNP from Man2 (via Ted1p in the ER) and from Man1 (by Cdc1p in the Golgi) functions as a quality assurance signal.

SUBMITTER: Yang G

PROVIDER: S-EPMC7927193 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Cdc1p is a Golgi-localized glycosylphosphatidylinositol-anchored protein remodelase.

Yang Gege G Banfield David K DK

Molecular biology of the cell 20201028 26

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive posttranslational modifications and remodeling, including the addition and subsequent removal of phosphoethanolamine (EtNP) from mannose 1 (Man1) and mannose 2 (Man2) of the glycan moiety. Removal of EtNP from Man1 is catalyzed by Cdc1p, an event that has previously been considered to occur in the endoplasmic reticulum (ER). We establish that Cdc1p is in fact a <i>cis</i>/medial Golgi membrane protein that relies on the C ...[more]

PMID: 33112703

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Project description:ObjectiveInvestigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).Materials and methodsPrimary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity.ResultsIn PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p < 0.05) and allogeneic (28% vs 58%; p < 0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha.ConclusionPIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.

Dataset Information

Cdc1p is a Golgi-localized glycosylphosphatidylinositol-anchored protein remodelase.

Publications

Cdc1p is a Golgi-localized glycosylphosphatidylinositol-anchored protein remodelase.

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