Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p

Abstract

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

FIGURE 1.

Two possible models for GPI biosynthetic pathway. A, if inositol acylation by Gwt1p occurs on the luminal side of the ER, GlcN-PI would flip from the cytoplasmic face to the luminal face of the ER membrane. B, if Arv1p is a flippase, GlcN-(acyl)PI, rather than GlcN-PI, would flip, and in this case, Gwt1p should function on the cytoplasmic face of the ER membrane.

FIGURE 2.

Sequence alignment of S. cerevisiae Gwt1p, mouse PIG-W, and their orthologs. Four regions that were highly homologous among 11 species are depicted. Identical amino acid residues among all 11 species are highlighted. Conservative residues among all 11 species are shaded. Positions that were mutated to alanine in S. cerevisiae Gwt1p are marked with asterisks above the sequences; positions mutated in mouse PIG-W are marked below the sequence. Sc, S. cerevisiae; Ca, C. albicans; Af, A. fumigatus; Nc, N. crassa; Cn, C. neoformans; Pf, P. falciparum; Ce, C. elegans; Ci, Ciona intestinalis; Xl, X. laevis; Hs, H. sapiens; Mm, M. musculus.

FIGURE 3.

Determination of essential residues of mouse PIG-W and S. cerevisiae Gwt1p. A, growth of GC1 cells transformed with mutant PIG-W. In this figure, “R133A,” for example, means that the arginine at the 133rd residue position of PIG-W was substituted with alanine. In GC1 cells, wild-type or mutant PIG-W was expressed under the control of TDH3 promoter from a YEp plasmid. Samples of each strain were precultured for 2 days in SG−Ura medium, diluted 1000-fold, cultured in SD−Ura at 30 °C for 2 days, and measured for absorbance at 620 nm. B, growth of GC1 cells transformed with wild-type or mutant forms of GWT1. The experimental procedure was the same as that described for A. Wild-type GWT1 was expressed under its own promoter (p_GWT1) or the TDH3 promoter (p_TDH3). All mutant GWT1 were expressed under the control of TDH3 promoter. Results on the upper and lower panels were obtained from different sets of experiments. C, growth of GC1 cells transformed with mutant S. cerevisiae GWT1 or mouse PIG-W on plates. The cells were spotted on SG−Ura (Galactose) or SD−Ura (Glucose) plates after making a 10× serial dilution (left to right) and then incubated at 30 °C for 2 days. M.m., M. musculus; S.c., S. cerevisiae. D, amount of mutant Gwt1p from GC1 cells was estimated by immunoblotting. Membrane fractions of the GC1 cells expressing wild-type or mutant Gwt1p were prepared as described under “Experimental Procedure” for glycosylation mapping. Each sample was split into two equal portions, which were separately subjected to SDS-PAGE. One gel (upper panel) was used for immunoblot analysis using anti-Gwt1p monoclonal antibody, and single and double asterisks indicate an intact and partially degraded Gwt1p, respectively. The other gel (lower panel) was subjected to Coomassie Brilliant Blue R-250 (CBB) staining as a loading control to assess relative protein content between samples.

FIGURE 4.

Prediction for the transmembrane domain of Gwt1p using three different prediction programs. TMpred, TMHMM, and SOSUI were used to identify putative transmembrane domains in Gwt1p. Shaded areas indicate putative transmembrane domains.

FIGURE 5.

Determination of membrane topology of Gwt1p by S2A glycosylation mapping. A, diagram of the positions of S2A sequence insertions. Shaded areas indicate putative transmembrane domains predicted by TMpred. Black numbers below arrows indicate the identification (ID) numbers of the fusion proteins in which Gwt1p remained functional after S2A insertion. Gray numbers below gray arrows indicate identification numbers in which Gwt1p lost its function after S2A insertion. B and C, immunoblot analysis of HA-tagged and S2A sequence-inserted Gwt1p, respectively. Endo-β-N-acetylglucosaminidase H (Endo H)-treated (+) and untreated (−) microsome fractions were subjected to SDS-PAGE and immunoblotting using an anti-HA monoclonal antibody. Identification numbers of the fusion proteins and their amino acid positions are shown above each lane. Gray identification numbers and amino acid positions in parentheses indicate that S2A sequence-inserted Gwt1p was not functional.

FIGURE 6.

Determination of membrane topology of Gwt1p by factor Xa protease cleavage analysis. Membrane fractions from GC1 cells expressing wild-type (WT) or Gwt1p fusion proteins with fXa cleavage sequences, tagged with an HA epitope, were prepared and digested with fXa in the presence (+) or absence (−) of 0.3% digitonin at 4 °C for 2 h. After digestion, digitonin-free samples were centrifuged and resuspended in fXa buffer. The samples were mixed with SDS-PAGE sample buffer and subjected to immunoblot analysis. An asterisk indicates intact Gwt1p detected with anti-HA antibody. Double asterisk indicates a partially degraded Gwt1p.

FIGURE 7.

Schematic diagram of the topology of Gwt1p. A, summary of the results of glycosylation (Glyco) mapping and fXa cleavage analysis. Four conserved regions (A–D) and essential residues (Asp-145, Lys-155, Asp-165, and Arg-216) are also shown. Asp-145 and Lys-155 were essential for both Gwt1p and PIG-W, but Asp-165 and Arg-216 were essential for PIG-W function alone. B, model of the membrane topology of Gwt1p. C, possible structure of four conserved regions. Numbers indicate amino acid positions.