The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p

Abstract

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.

Figure 1

Blue native PAGE of microsomal membrane proteins during Gpi8p depletion. FBY164 (gpi8::kanMX2 ura3–1:: URA3-GAL1,10-GPI8) cells were grown overnight at 24°C in SGaa medium, were shifted to SDaa medium, and were incubated for 0, 4, 7, and 23 h at 37°C. After incubation, the cells were harvested, microsomes were prepared, and membrane proteins were solubilized in digitonin and processed for blue native PAGE. Solubilized proteins (40 μg per lane) were loaded on a 5–15% polyacrylamide gradient gel. Proteins were transferred onto a PVDF membrane and probed with antibodies against Gpi8p. Marker proteins were bovine serum albumin (monomeric and dimeric form, 66 and 132 kDa, respectively), apoferritin (443 kDa), and thyroglobulin (660 kDa).

Figure 2

Affinity chromatography purification of a multiprotein complex containing the GST-tagged form of Gpi8p. (A) Membranes of FBY656 cells (gpi8Δ GST-Gpi8p) were isolated and solubilized either in the presence (+) or absence (-) of 6-aminocaproic acid (aca isol). The bulk of the lysate prepared without 6-aminocaproic acid was subjected to affinity chromatography on glutathione-Sepharose 4B. Solubilized proteins were prepared for electrophoresis in the loading buffer with (+) or without (-) 6-aminocaproic acid (aca migr). Aliquots from extracts (lanes 1–4) and from affinity-purified protein (lanes 5 and 6) were separated on a 5–12% blue native gel. Gpi8p was detected by Western blotting with anti-Gpi8p antibody. (B) Material bound to glutathione-Sepharose was eluted with the use of 20 mM, then 100 mM, reduced glutathione (eluates 20 and 100, respectively). The crude extract (lane 1), material not binding to the Sepharose (lane 2), washes 1 and 3 (lanes 3 and 4), and eluates (lanes 5 and 6) were loaded onto a 12% polyacrylamide gel for SDS-PAGE. The gel was stained with silver nitrate. (C) The presence of the GST-tagged form of Gpi8p in the eluates was confirmed by probing a Western blot of a parallel gel containing the same fractions with anti-Gpi8p antibodies. The amounts of material loaded onto the gels are indicated in OD₆₀₀ equivalents.

Figure 3

Mass spectrometric identification of Gpi8p-associated proteins. (A) Spectrogram of peptide masses obtained from tryptic digestion of bands a and b (analyzed jointly) from the gel shown in Figure 2B, lanes 5 and 6. Peptide masses were used to query an amino acid sequence database (PROTEIN PROSPECTOR). Proteins were identified by correlating the measured peptide masses to a theoretical tryptic digest of all proteins present in the database. Yeast Gpi8p and Gpi16p were the only yeast proteins that were identified. Their peptides are marked with crosses (+, Gpi8p) and dots (●, Gpi16p). (B) Spectrogram of peptide masses obtained from band c (Figure 2B) identified Gaa1p (*). The exact tryptic peptides allowing identification are listed. a.i. = absolute intensity.

Figure 4

Sequence of S. cerevisiae Gpi16p. (A) Potential N-glycosylation sites are shown in boldface, the C-terminal TMD is underlined, and the ER retrieval domain doubly underlined. The most likely cleavage site of the signal peptidase is indicated by a vertical arrow. The Gpi16p homologues of Schizosaccharomyces pombe (emb CAA22348.1 ), Drosophila melanogaster (gb AAF46367.1 ), Homo sapiens (CGI-06 protein, XM 009533.2), Caenorhabditis elegans (emb CAA96629.1), and Arabidopsis thaliana (gb AAF20232.1 ) were aligned usig the CLUSTALW multiple sequence alignments program 1.81. Only the yeast Gpi16p sequence is shown, but identities and conservative substitutions are represented by black and gray boxes. (B) A hydropathy plot, drawn according to Kyte and Doolittle, is shown in which the hydrophobic sequences get a positive score.

Figure 5

Cells depleted of Gpi16p accumulate complete GPI lipids. (A) W303 and FBY735 (gpi16Δ YCplac111-GAL1-GPI16) were grown to exponential phase at 30°C in SGaa. The expression of Gpi16p was subsequently blocked by shifting cells to glucose medium (SDaa). Cultures were continued at 30°C for 0, 5, 15, or 40 h and were periodically diluted with fresh medium to keep the cell concentration in the range of 0.5–2.5 OD₆₀₀. The sec18 gpi8 cells (lane 1) were grown at 24°C and were preincubated for 5 min at 37°C. Aliquots of cells were radiolabeled at 37°C with [2-³H]-myo-inositol for 80 min, and lipid extracts were analyzed by TLC. (The sec18 gpi8 double mutant was chosen as a positive control, since it accumulates more CP2 at 37°C than gpi8.) (B) FBY733b cells (gpi16Δ pYES2-GPI16) were grown to exponential phase at 30°C in SGaa medium and were shifted to SDaa for 2 h (lane 3) or 15 h (lane 4). W303 cells and yjr015Δ cells were grown at 30°C. Aliquots of cells were radiolabeled and processed as above.

Figure 6

Depletion of Gpi16p blocks maturation of Gas1p and Cwp1p. (A) W303–1B and FBY735 cells (gpi16Δ YCp-GAL1-GPI16) were grown to exponential phase at 30°C in SGaa medium and were shifted to glucose for 0, 5, and 15 h in order to block the expression of Gpi16p. Proteins were extracted, separated by SDS-PAGE, Western blotted, and probed with anti-Gas1p and anti-Cwp1p antibodies. The relative amounts of the immature 105-kDa form of Gas1p as well as of the immature 45-kDa form of Cwp1p were determined by densitometry and are given as the percentage of total Gas1p or Cwp1p in the extracts. m = mature; i = immature. (B) FBY735 cells were grown to exponential phase at 30°C in selective SGaa medium, were washed twice in distilled water, and were resuspended in SDaa medium. After 0 or 15 h of culture, cells were pulse-labeled with [³⁵S]-methionine and [³⁵S]-cysteine for 10 min and were chased for the indicated times. Gas1p was immunoprecipitated from the lysates and analyzed by SDS-PAGE and fluorography.

Figure 7

Gpi16p is a lumenally oriented, integral membrane glycoprotein of the ER. (A) Exponentially growing W303–1B were broken with glass beads and cell walls were removed. Aliquots of the lysate (equivalent to 20 OD₆₀₀) were incubated for 30 min at 0°C with NaCl, Na₂CO₃, Triton X-100, or SDS. Subsequently, membranes were sedimented by ultracentrifugation, proteins of supernatants (S) and pellets (P) were precipitated with TCA, were processed for SDS-PAGE and Western blotting, and were probed with affinity-purified rabbit anti-Gpi16p antibodies. (B) Microsomes from W303–1B were digested with 0.1 mg/ml proteinase K at 0°C for 30 min in the presence or absence of 0.5% of Triton X-100. Samples were resuspended in sample buffer, processed for Western blotting, and probed with anti-Gpi16p or anti-Gpi8p antibodies. (C) A microsomal pellet from W303–1B cells was solubilized in 1% SDS and was incubated with or without 1 mU/OD₆₀₀ endoglycosidase H for 20 h at 37°C. The samples were processed as above. (D) Cellular localization of Gpi16p was determined by subcellular fractionation. For this experiment, a slightly modified version of a published procedure (Gaynor et al., 1994) was used. Spheroplasts of W303–1B at 67 OD₆₀₀/ml were osmotically lysed in a syringe, the pellet P13 obtained through centrifugation at 13,000 × g for 15 min was further resolved by sedimenting 100-OD₆₀₀ equivalents through a step gradient of 1.6 ml 1.2 M sucrose to >1.6 ml 1.5 M sucrose, yielding 5 0.7-ml fractions termed F1 to F5. Supernatant S13 was further centrifuged at 100,000 × g for 1 h to obtain pellet P100. Equivalent aliquots of all fractions were processed for SDS-PAGE and Western blotting. On prolonged incubation of microsomes, variable amounts of a 69-kDa degradation product of Gpi16p was generated.

Figure 8

Gpi8p and Gpi16p are mutually stabilizing each other. FBY164 (gpi8::kanMX2 ura3–1:: URA3-GAL1,10-GPI8), W303, and FBY735 (YHR188c::kanMX4 containing plasmid YCplac111-GAL1-GPI16) were grown to exponential phase at 30°C in galactose medium (SGaa). The expression of Gpi8p (FBY164) or Gpi16p (FBY735) was subsequently blocked by shifting cells to glucose medium (SDaa). Cultures were continued at 30°C for 0, 4, 7, and 22 h (FBY164) or for 0, 5, 15, and 40 h (W303 and FBY735). Proteins were extracted, separated by SDS-PAGE, Western blotted, and probed with anti-Gpi8p (A) and anti-Gpi16p antibodies (B); membranes were stripped and probed again with anti-Gpi16p (A) and anti-Gpi8p (B) antibodies.

Figure 9

Overexpression of Gpi8p cannot suppress the growth defect of Gpi16p-depleted cells. (A) W303–1B (wt), FBY735 (Δgpi16/YCplac111-GAL1-GPI16), FBY735 transfected either with empty YEplac195, or with YEplac195 harboring GPI8 placed under the control of the promoter of CUP1 (YEplac195-CUP1-GPI8) were grown to exponential phase at 30°C in galactose-containing medium. To deplete them of Gpi16p and to induce Gpi8p, 3-μl aliquots of cells at concentrations shown at the top (cells/ml) were inoculated onto plates containing glucose plus 100 μM Cu²⁺. The plates were incubated for 35 h at 30°C before being photographed. (B) W303 (lanes 1 and 5), FBY735 (lanes 2 and 6), FBY 735 transfected with YEplac195-CUP1-GPI8 (lanes 3 and 7), and FBY735 transfected with the empty vector YEplac195 (lanes 4 and 8) were grown at 30°C for 15 h in liquid culture with the use of either galactose medium or, alternatively, glucose medium supplemented with 100 μM Cu²⁺. Proteins were extracted, separated by SDS-PAGE, Western blotted, and probed with anti-Gpi8p antibodies.

Figure 10

Association of the transamidase complex with other multimeric ER complexes. The membrane of the Western blot shown in Figure 2A was stripped and probed again with anti-Sec61p antibodies, was stripped again, and finally was probed with anti-Wbp1p antibodies. The top panel is the same as in Figure 2A.

Figure 11

Stability of the transamidase complex in the absence of GPI protein and GPI lipid precursors. (A) W303 cells were grown to exponential phase at 37°C in YPD medium. Cycloheximide (Chx; 100 μg/ml) was added 0, 15, or 60 min before protein extraction. Thermosensitive mutant cells gpi1, gaa1–1, and mcd4 were grown to exponential phase at 24°C in YPD medium. The cells were left at 24°C (-) or were shifted to 37°C for 15 or 60 min. Then, microsomal membranes were prepared and proteins were processed for blue native PAGE. Five μg per lane of solubilized proteins was loaded on a 5–15% gradient gel. Proteins were transferred onto a PVDF membrane and were probed with antibodies against Gpi8p. (B) Five micrograms of the same proteins from W303 and gpi1 were separated on SDS-PAGE (7.5%). Mature (m) and immature (i) forms of Gas1p were detected by Western blotting with the use of rabbit anti-Gas1p antibodies. The percentage of immature Gas1p, as quantitated by densitometry, is indicated below. Similarly to the original gpi1 isolate and to gpi1Δ cells (Leidich et al., 1994; Leidich and Orlean, 1996), the gpi1 mutant used here neither grows nor incorporates [2-³H]-myo-inositol into proteins at 37°C, whereas at 24°C GPI anchoring is only partially deficient. This partial deficiency explains the abnormally high percentage of immature Gas1p in gpi1 already at 24°C.