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.

FIGURE 1:

Cdc1p is a Golgi-localized membrane protein. (A) Abbreviated depiction of the remodeling of GPI-APs in the yeast ER. (B) The steady-state levels of Gas1p in cells lacking the indicated ER resident GPI-AP remodelases (see A). mGas1p and pGas1p denote the mature (plasma membrane) and precursor (ER) forms of Gas1p, respectively. Pgk1p serves as a protein loading control. (C) mNeon-CDC1 and CDC1-mNeon are functional. The endogenous CDC1 gene was replaced with a version of the gene in which the protein coding sequence of mNeon was inserted at either the 5′ (mNeon-CDC1) or 3′ (CDC1-mNeon) end of the gene. Tenfold serial dilutions of the indicated strains were spotted onto YEPD plates with or without 50 µg/mL CFW. (D) mNeon-Cdc1p partially colocalizes with the cis Golgi resident protein Mnn9p (∼60%) and the medial Golgi resident protein Kre2p (∼40%). See Materials and Methods for further details. (E) Cdc1p-mNeon partially Cdc1p partially colocalizes with the cis Golgi resident protein Mnn9p (∼60%) and the medial Golgi resident protein Kre2p (∼40%). See Materials and Methods for further details. Arrowheads denote colocalizing puncta.

FIGURE 2:

Cdc1p does not cycle between the Golgi and ER but relies on COPI for its Golgi retention. (A) Unlike mCherry-Sed5p, mNeon-Cdc1p does not cycle between the Golgi and the ER. Arrowheads indicate the ER membranes that envelop the nucleus. (B) In the COPI-coatomer mutants sec21-1 (which encodes γ-COP) and sec27-1 (which encodes β’-COP; see Supplemental Table S1), mNeon-Cdc1p is mislocalized to the vacuole. (C) Cdc1-mNeon remains in the Golgi in cells lacking VPS74 (vps74Δ) or BRE5 (bre5Δ); see text for details. (D) In vitro mixing assays assessing the interaction between the cytoplasmic regions of Cdc1p and COPI-coatomer. WCEs from WT cells were mixed with the indicated bacterially expressed purified Cdc1p peptides and binding assessed by SDS–PAGE and immune staining. GST-KKXX serves as a positive control. (E, F) In vitro mixing assays assessing the interaction between the cytoplasmic regions of Cdc1p and the COPII protein Sec24p. WCEs from cells expressing myc-tagged Sec24p (see Supplemental Table S1) were mixed with the indicated bacterially expressed purified Cdc1p peptides and binding assessed by SDS–PAGE and immune staining. Sed5-GST serves as a positive control (see Supplemental Table S2) (G) mNeon-Cdc1p Golgi localization is unaffected in erv14Δ cells. (H) mNeon-Cdc1p Golgi localization is unaffected in emp24Δ cells, while mNeon-GPI ER export is delayed. IB denotes the primary antibodies used.

FIGURE 3:

CDC1 temperature-sensitive mutants exhibit cell wall defects and are deficient in the transport of a GPI-anchored protein to the cell surface. (A) cdc1 (L356F) and cdc1 (T377I) cells are not ts for growth. Tenfold serial dilutions of the indicated strains were spotted onto YEPD plates and incubated at 25 and 37°C for 48 h before being photographed. (B) The distribution of amino acid substitutions in the cdc1-21p–cdc1-26p transmembrane domain. (C) The temperature sensitivity of cdc1-21–cdc1-26 alleles is osmoremedial. Tenfold serial dilutions of the indicated strains were spotted onto YEPD plates with or without 1M sorbitol and incubated at 25 and 37°C for 48 h before being photographed. (D) cdc1-21–cdc1-26 cells show increased sensitivity to CFW. Tenfold serial dilutions of the indicated strains were spotted onto YEPD plates with or without 50 µg/mL CFW and incubated at 25°C for 48 h before being photographed. (E) At 37°C ∼40% of α-factor-treated cdc1-23 and cdc1-26 cells arrest growth with polarization defects and small buds. White arrowheads denote the small bud phenotype and the star symbol denotes a polarization anomaly. The quantification data are derived from three separate experiments in which at least 100 cells were assessed for each strain. (F) cdc1-21–cdc1-26 cells do not accumulate ER or hypoglycosylated forms of Gas1p at the nonpermissive temperature for growth (37°C). Equivalent amounts of WCEs from the indicated strains (grown at 25 and 37°C) were resolved by SDS–PAGE and Gas1p was detected by immune staining with an anti-Gas1p antibody. The mature form of Gas1p (mGas1p) is indicated with an arrow. Pgk1p serves as a gel loading control. (G) mNeon-Gas1p is localized to the cell surface, vacuole, and internal structures in cdc1-22 and cdc1-26 cells grown at 25°C. (H) cdc1-23 and cdc1-26 cells accumulate ∼25–30% more Proteinase K–resistant Gas1p than WT cells when grown at 37°C. (I) Quantification of the data shown in H; see Materials and Methods for details.

FIGURE 4:

Cdc1p’s enzymatic activity is restricted to the Golgi. (A) cdc1 ted1Δ cells show an osmo-remedial synthetic temperature-sensitive phenotype. Tenfold serial dilutions of the indicated strains were spotted onto YEPD plates with or without 1M sorbitol and incubated at 25 and 37°C for 48 h before being photographed. (B) mNeon-Cdc1-HDEL is localized to the ER in cells that concomitantly overexpress Erd2p (2μ ERD2). White arrowheads indicate ER membranes. (C) Cells expressing endogenous levels of mNeon-Cdc1-HDEL that concomitantly overexpress Erd2p (2μ ERD2) cannot support the growth cdc1Δ cells. Tenfold serial dilutions of the indicated strains were spotted onto plates with or without 1 mg/ml 5-FOA and incubated at 25°C for 72 h before being photographed. (D) When overexpressed, mNeon-Cdc1-HDEL is localized to the ER and puncta. (E) When overexpressed, mNeon-Cdc1-HDEL can support the growth cdc1Δ cells. Tenfold serial dilutions of the indicated strains were spotted onto SD-Leu plates and incubated at 25 and 37°C for 48 h before being photographed. (F) ER-localized Cdc1p does not have a dominant negative effect on the processing of Gas1p. Cells expressing mNeon-Cdc1-HDEL from the TPI promoter in the presence of a 2μ ERD2 plasmid (ER-localized Cdc1p) do not show a synthetic accumulation of the precursor form of Gas1p (pGas1p). WCEs from the indicated strains (including two isolates of mNeon-Cdc1p-HDEL + 2μ ERD2-expressing cells) were resolved by SDS–PAGE and Gas1p detected by immune staining with an anti-Gas1p antibody. The mature form (mGas1p) and precursor form (pGas1p) are indicated with arrows. Pgk1p serves as a gel loading control. The presence of pGas1p detected in each strain is represented graphically below the immune-staining experiment. White arrowheads indicate ER membranes.