©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Temperature-sensitive Yeast GPI Anchoring Mutants gpi2 and gpi3 Are Defective in the Synthesis of N-Acetylglucosaminyl Phosphatidylinositol
CLONING OF THE GPI2 GENE (*)

Steven D. Leidich (1), Zlatka Kostova (1), Robert R. Latek (1), Lisa C. Costello (1), Darren A. Drapp (1), William Gray (2), Jan S. Fassler (2), Peter Orlean (1)(§)

From the (1) Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 and the (2) Department of Biology, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To identify genes required for the synthesis of glycosyl phosphatidylinositol (GPI) membrane anchors in yeast, we devised a way to isolate GPI anchoring mutants in which colonies are screened for defects in [H]-inositol incorporation into protein. The gpi1 mutant, identified in this way, is temperature sensitive for growth and defective in vitro in the synthesis of GlcNAc-phosphatidylinositol, the first intermediate in GPI biosynthesis (Leidich, S. D., Drapp, D. A., and Orlean, P.(1994) J. Biol. Chem. 269, 10193-10196). We report the isolation of two more conditionally lethal mutants, gpi2 and gpi3, which, like gpi1, have a temperature-sensitive defect in the incorporation of [H]inositol into protein and which lack in vitro GlcNAc-phosphatidylinositol synthetic activity. Haploid Saccharomyces cerevisiae strains containing any pairwise combination of the gpi1, gpi2, and gpi3 mutations are inviable. The GPI2 gene, cloned by complementation of the gpi2 mutant's temperature sensitivity, encodes a hydrophobic 269-amino acid protein that resembles no other gene product known to participate in GPI assembly. Gene disruption experiments show that GPI2 is required for vegetative growth. Overexpression of the GPI2 gene causes partial suppression of the gpi1 mutant's temperature sensitivity, a result that suggests that the Gpi1 and Gpi2 proteins interact with one another in vivo. The gpi3 mutant is defective in the SPT14 gene, which encodes a yeast protein similar to the product of the mammalian PIG-A gene, which complements a GlcNAc-phosphatidylinositol synthesis-defective human cell line. In yeast, at least three gene products are required for the first step in GPI synthesis, as is the case in mammalian cells, and utilization of several different proteins at this stage is therefore likely to be a general characteristic of the GPI synthetic pathway.


INTRODUCTION

Diverse proteins in eukaryotic cells are anchored in the external face of the plasma membrane by a GPI() anchor (1, 2) . GPIs are assembled in the membrane of the endoplasmic reticulum, then transferred to protein in the lumen of that organelle, after which the GPI-anchored proteins are transported to the cell surface via the secretory pathway. GPIs have been proposed to be a signal in protein targeting (3) , to participate in signal transduction by promoting clustering of the receptor proteins they anchor with other proteins in trans-membrane signaling pathways (4, 5) , and to serve as intermediates in the transfer of glycoproteins from the yeast plasma membrane to acceptor glycans in the cell wall (6) .

Studies of GPI anchoring-deficient cells should shed light on both the function and synthesis of GPI anchors. A number of mutant mammalian cell lines have been isolated that fail to express GPI-anchored proteins on their cell surface (7, 8, 9, 10, 11, 12) , and cells from patients with paroxysmal nocturnal hemoglobinuria are also GPI anchoring defective (13-15). These mutant cell lines include three complementation groups that are defective in GlcNAc-PI synthesis, namely mutants defective in the PIG-A and GPI-H genes, as well the Thy-1 Class C murine lymphoma mutant (14, 16, 17) .

We set out to explore the synthesis and function of GPI anchors in genetically tractable eukaryotic microbes and developed a way to identify yeast GPI anchoring mutants in which colonies are screened for those defective in the incorporation of [H]inositol into protein (18) . The gpi1 mutant, which was identified in this way, is temperature sensitive both for growth and for [H]inositol incorporation into protein and lacks the in vitro activity that catalyzes the first step in GPI synthesis, the formation of GlcNAc-PI. Our finding that the gpi1 mutant was temperature sensitive prompted us to screen a collection of temperature-sensitive yeast mutants for further strains defective in [H]inositol incorporation into protein, hence in GPI anchoring. We report here the isolation of two further mutants, gpi2 and gpi3, which are defective in vitro in the synthesis of GlcNAc-PI. The GPI2 gene encodes a protein that does not resemble any protein known so far to participate in GPI synthesis, whereas the gpi3 mutant is defective in the yeast counterpart of PIG-A (19, 20), a human gene that complements a human GlcNAc-PI synthesis-deficient cell line.


EXPERIMENTAL PROCEDURES

Yeast Strains and Culture Media

The wild type Saccharomyces cerevisiae strains used were PP-1B (his4 chs1::URA3 ura3-52 MATa) (18) , XM37-10C (ade2-101 ura3-52 MAT ) (18), and 9933-13A (his3 ura3 leu2 trp1 MATa) (23) . gpi1-8A (gpi1 MAT ade2-101 ura3-52 his4) was a segregant of the fourth outcross of the gpi1 mutation (18) . Strain JF1015 (spt14-3 ura3-52 leu2 lys2 his4-917), allelic to gpi3, is described in Ref. 19. The bank of temperature-sensitive lethal S. cerevisiae mutants isolated by Vijayraghavan et al.(21) was obtained from the laboratory of J. Abelson. The primary isolates of the two GPI anchoring-deficient mutants identified in this collection and studied further were SS328-27 and SS328-53, whose genotype was MAT ade2-101 his3 200 lys2-801 ura3-52. These strains harbor, respectively, the gpi2 and gpi3 mutations. Strains 27-5A (gpi2 ura3-52 MATa), 27-5D (gpi2 ade2 ura3-52 MATa), and 53-12C (gpi3, lys2 MATa) were segregants from the second outcross of the gpi2 and gpi3 mutations. Strain 27-1C (gpi2 ura3-52 MATa) was a segregant from the third outcross of the gpi2 mutation and used for integrative mapping of the GPI2 locus.

Growth media and materials were as described in Ref. 18. Synthetic complete medium was prepared as described in Ref. 22. Screen for Defects in [H]Inositol Incorporation into Protein-Patches of cells grown at 25 °C on inositol-free synthetic medium were lifted onto filter paper replicas, shifted to 42 °C for 20 min, then pulse labeled with [H]inositol for 40 min at 42 °C as detailed in Ref. 18. The filter-bound, radiolabeled cell patches were then extracted with ethanol/water/diethylether/pyridine/NHOH (15:15:5:1:0.018 by volume) to remove all background signal due to [H]inositol-labeled lipids. Remaining protein-linked [H]inositol was then detected by fluorography. [H]Inositol Labeling of Protein and Lipids-[H]Inositol labeling of protein and lipids for subsequent separation, respectively, by SDS-polyacrylamide gel electrophoresis and thin layer chromatography was carried out as detailed in Ref. 18.

Enzyme Assays

Twice-washed mixed membranes were prepared from cultures of wild type and mutant cells that had been grown at 25 °C (23) . Synthesis of [C]GlcNAc-PI, [C]GlcN-PI, Dol-PP-[C]GlcNAc, and Dol-P-[C]Man and de-N-acetylation of [C]GlcNAc-PI were assayed as described in Ref. 18. Palmitoyl CoA-dependent acylation of inositol in GlcN-PI was assayed by incubating membranes with TLC-purified [C]GlcN-PI and 0.14 mM palmitoyl CoA (23) .

Cloning of the GPI2 Gene

A yeast genomic DNA library constructed in the centromeric plasmid YCp50 (24) was introduced into the gpi2 ura3 strain 27-5D using the lithium acetate transformation protocol (22) . Uracil-prototrophic transformants were selected at 25 °C, some 30,000 being obtained. When colonies of transformants just became visible (after 24-30 h of growth), the agar plates spread with the transformants were shifted to 37 °C, and incubation was continued at this temperature. Plasmids were isolated from those colonies that continued to grow at 37 °C.

Molecular Biological Techniques and DNA Sequence Analysis

Standard procedures were used for the propagation and selection of plasmids and the growth of their bacterial hosts and for the subcloning of DNA fragments (25) . To locate the GPI2-complementing DNA sequence, restriction fragments of complementing plasmid pGC2-8.5 were subcloned into the yeast 2 micron shuttle vector pRS426 (26) . Cells of gpi2 ura3 strain 27-5D transformed with plasmids containing these different restriction fragments were tested for restoration of their ability to grow at 37 °C.

DNA sequencing was carried out using the dideoxy chain termination method (27) according to the double-stranded sequencing protocol described in the U. S. Biochemicals Sequenase version 2.0 sequencing kit. DNA fragments cloned into plasmid pRS426 were used as templates. Oligonucleotide primers were synthesized by the Genetic Engineering Facility (University of Illinois at Urbana-Champaign). Analyses of DNA and protein sequence data were performed using the DNA Strider 1.1 program (28) .

For Southern blot analyses, total chromosomal DNA was isolated from the untransformed Ura diploid and from two Ura transformants, essentially as described in Ref. 22. DNA was digested with HindIII (6 units/µg), electrophoresed through 1% agarose, and transferred (25) to nylon membranes (Boehringer Mannheim). DNA was cross-linked to the membrane using a UV Stratalinker 1800 (Stratagene) delivering 120,000 µJ of ultraviolet radiation. The probe used was a 1.4-kilobase HindIII-SalI fragment containing the COOH-terminal half of the GPI2 gene, which was labeled with digoxigenin-dUTP using the Genius random-primed DNA labeling and detection kit (Boehringer Mannheim). Membranes were prehybridized in a rotating oven (National Labnet Co. Woodbridge. NJ) at 65 °C for 2 h with 25 ml of 5 SSC containing 0.1% (v/v) N-lauroylsarcosine, 0.02% (w/v) SDS, and 1.0% blocking reagent. Digoxigenin-labeled probe was added to the prehybridization solution at a concentration of approximately 30 ng/ml and was allowed to hybridize to the membranes overnight at 65 °C. Membranes were washed twice with 2 SSC containing 0.1% SDS for 10 min at 65 °C and then twice with 0.5 SSC, 0.1% SDS for 15 min at 65 °C. Hybridized DNA fragments were detected using anti-digoxigenin antiserum and Lumi-Phos 530, in accordance with the manufacturer's instructions (Boehringer Mannheim).

Disruption of the GPI2 Gene

The transformation technique (29) was used to disrupt the GPI2 gene. An EcoRI-SalI fragment containing the 5`-portion of the GPI2 gene and a HincII-EcoRI fragment encoding the GPI2 3`-sequence were subcloned in tandem, but in reverse order, in the SalI and SmaI sites of the yeast-integrating vector pRS406 (29) , creating a unique EcoRI site between the two segments of GPI2 sequence (Fig. 6A). The EcoRI site in the HincII-EcoRI fragment is derived from polylinker sequence in the plasmid from which the fragment was excised. Construction of this plasmid, pgpi2::URA3, resulted in the deletion of a 167-base pair SalI-HincII portion of the coding region of GPI2. Plasmid pgpi2::URA3 was linearized by digestion with EcoRI and used to transform the Ura diploid strain 9933-13A XM37-10C. Ura transformants were selected, and two independent Ura diploids were analyzed by Southern blotting to confirm that the disrupted copy of GPI2 had been integrated at the expected chromosomal locus.


Figure 6: Disruption of the GPI2 gene. A, construction of plasmid pgpi2::URA3 used to disrupt the GPI2 gene following the transformation procedure (29). B, Southern blot analysis to confirm integration of pgpi2::URA3 at the chromosomal GPI2 locus. Total chromosomal DNA was isolated from the wild type Ura- untransformed diploid strain 9933-13A XM37-10C and from two Ura+ GPI2/gpi2::URA3 diploid transformants and digested with HindIII. Following agarose gel electrophoresis and transfer to a nylon membrane, the DNA was probed with the digoxigenin-labeled probe that contains the 3`-portion of the GPI2 gene. The expected sizes of the bands yielded by the HindIII digest are 3.8 (wild type (WT)) and 8.0 kilobases (kb) (gpi2::URA3 disruption).




RESULTS

Biochemical Characterization of Two New GPI Anchoring Mutants

Since the GlcNAc-PI synthesis-defective gpi1 mutant was temperature sensitive for growth, we reasoned that mutations in further genes encoding proteins involved in GPI synthesis could also give rise to conditional lethality. We therefore screened patches of cells from a bank of 940 temperature-sensitive mutants (21) for further strains defective in the incorporation of [H]inositol into protein (18) . We focus here on two new GPI anchoring mutants we have identified. Biochemical analyses were carried out on the primary isolates, strains SS328-27 and SS328-53, and on haploid segregants obtained after the second outcross of these mutants to wild type strains (strains 27-5A and 53-12C).

Mutants 27-5A and 53-12C showed a temperature-sensitive defect in [H]inositol incorporation into protein, as did gpi1 (Fig. 1). Analysis of lipids radiolabeled with [H]inositol at 25 and 38 °C showed that strains 27-5A and 53-12C, like gpi1, incorporated substantial amounts of [H]inositol into P[H]I and are therefore affected in neither the uptake of [H]inositol nor in the synthesis of P[H]I, the immediate precursor of GPIs (Fig. 2). In all three mutants, incubation at high temperature had a similar effect on certain lipids that are not GPI precursors; incorporation of [H]inositol into lyso-PIs and mannosyl di-inositol phosphoceramide was lowered. However, [H]inositol incorporation into mannosyl di-inositol phosphoceramide was also perturbed at high temperature in the wild type strain, and formation of mannosyl inositol phosphoceramide, the precursor of mannosyl di-inositol phosphoceramide, was not affected in the mutants. These perturbations in the synthesis of certain [H]inositol-labeled lipids may therefore be a secondary effect of the temperature shift and cessation of growth of the mutants.


Figure 1: Three yeast mutants with a temperature-sensitive block in [H]inositol incorporation into protein. Cultures of gpi1-8A, 27-5A (gpi2), and 53-12-C (gpi3) were grown at 25 °C and divided into two equal portions, one of which was shifted to 38 °C for 20 min. The shifted and control cultures were then radiolabeled with 100 µCi of [H]inositol/ml for 40 min at 38 and 25 °C, respectively, after which radiolabeled proteins were extracted and separated by SDS-polyacrylamide gel electrophoresis, and H-labeled proteins were made visible by fluorography. WT, wild type.




Figure 2: Normal uptake of [H]inositol and synthesis of P[H]I by the three yeast GPI anchoring mutants. Strains gpi1-8A, 27-5A (gpi2), and 53-12-C (gpi3) were radiolabeled with 30 µCi of [H]inositol/ml at 25 and 38 °C as described for Fig. 1. Radiolabeled lipids were then extracted with ethanol/water/diethylether/pyridine/NHOH (15:15:5:1:0.018 by volume), separated by thin layer chromatography, and then made visible by fluorography, all as described in Ref. 18. Identities of [H]inositol-labeled lipids were assigned according to Refs. 38 and 39. IPC, inositol phosphoceramide; MIPC, mannosyl inositol phosphoceramide; M(IP)C, mannosyl di-inositol phosphoceramide.



Neither mutant 27-5A nor 53-12C showed any accumulation of a potential [H]inositol-labeled GPI precursor at non-permissive temperature. To establish whether the mutants have a specific defect early in the GPI synthetic pathway, we assayed for in vitro GPI biosynthetic activities. Membranes from the two candidate mutants were incubated with UDP-[C]GlcNAc, and radiolabeled lipids were extracted and separated by TLC. Whereas wild type membranes synthesized the first two yeast GPI precursors, [C]GlcNAc-PI and [C]GlcN-PI (18, 21) (Fig. 3, lanes1 and 5), membranes from isolates SS328-27 and SS328-53 made neither (Fig. 3, lanes3, 4, 7, and 8) and therefore, like the control gpi1 membranes (Fig. 3, lanes2 and 6), are defective in GlcNAc-PI synthesis.


Figure 3: Mutant membranes lack in vitro GlcNAc-PI synthetic activity but show enhanced synthesis of Dol-PP-GlcNAc. Washed mixed membranes were prepared from wild type strain PP-1B and from mutant strains gpi1-8A, SS328-27, and SS328-53, which had all been grown at 25 °C. Glycosyltransferase assays and subsequent thin layer chromatographic separation of radiolabeled lipids were performed as detailed in Refs. 18 and 22. The same results were obtained using strains 27-5A and 53-12-C, segregants from the second out-cross of the original mutant strains. Lanes 1-4, assay for synthesis of [C]GlcNAc-PI and [C]GlcN-PI. Lanes 5-8, assay for the synthesis of Dol-PP-[C]GlcNAc. Tunicamycin (TM) was left out of, and Dol-P added to, these incubations. Lanes 9-12, synthesis of Dol-P-[C]Man. WT, g1, 27, and 53, wild type, gpi1-8A, SS328-27, and SS328-53 strains, respectively.



We have found with gpi1 membranes that in the absence of GlcNAc-PI synthetic activity, much more Dol-PP-[C]GlcNAc are made (18) . A straightforward explanation of this is that more UDP-[C]GlcNAc is available to the first two enzymes of the asparagine-linked glycosylation pathway, which also use this sugar nucleotide as donor. When membranes from isolates SS328-27 and SS328-53 were incubated with UDP-[C]GlcNAc in the absence of tunicamycin, they, like gpi1 membranes, made larger amounts of Dol-PP-GlcNAc ( Fig. 3lanes6-8). This result indicates that the defect in the two new mutants is indeed in UDP-GlcNAc utilization.

Mutant membranes were also tested for their ability to carry out the next two steps in the GPI synthetic pathway, deacetylation of GlcNAc-PI to GlcN-PI and then acylation of GlcN-PI to yield GlcN-(acylinositol)PI; both activities were present in the mutant membranes (not shown). Levels of activity of Dol-P-Man synthase, a glycosyltransferase of the endoplasmic reticulum that is required for GPI synthesis in yeast in vivo(30) , were similar in mutant and wild type membranes (Fig. 3, lanes 9-12).

Genetic Characterization of the Mutants

In crosses of isolates SS328-27 and SS328-53 to wild type strains, it was established that the mutations in these strains were recessive and that the [H]inositol-labeling defect and temperature sensitivity for growth cosegregated in three successive back-crosses. Further crosses showed that the two new GPI anchoring mutants, SS328-27 and SS328-53, and the gpi1 mutant are in separate linkage groups. Mutants SS328-27 and SS328-53 will henceforth be designated gpi2 and gpi3, respectively. The temperature sensitivity of the three mutants could not be suppressed upon increasing the osmolarity of the medium by supplementation with either 0.25 M KCl or 1 M sorbitol; the GPI anchoring mutants are therefore not osmotically remedial.

Haploid strains containing pairs of GPI anchoring mutations are not viable. gpi1, gpi2, and gpi3 haploids were mated to one another, and the resulting gpi1/gpi2, gpi1/gpi3, and gpi2/gpi3 diploids were induced to sporulate. Asci from 23 gpi1/gpi2, 21 gpi1/gpi3, and 20 gpi2/gpi3 diploids were dissected and allowed to germinate at 25 °C on YPD medium. The patterns of viability of the haploid segregants and of the segregation of temperature sensitivity among the viable haploids were diagnostic of double mutant lethality. Thus, where tetrads yielded two viable and two non-viable haploids, both viable haploids grew at 37 °C. Where tetrads yielded three viable and one inviable haploid, one segregant grew at 37 °C, and the other two were temperature sensitive for growth. Where all four haploid segregants were viable at 25 °C, they were all temperature sensitive. The same results were obtained when the ascospores were dissected onto YPD medium supplemented with either 0.25 M KCl or with 1 M sorbitol; the double gpi mutants are therefore not osmotically remedial at 25 °C.

The gpi3 Mutant Is Defective in the Yeast Counterpart of the Human PIG-A Gene

The gpi3 mutant proved to be in the same complementation group as the spt14 mutant, which had previously been isolated in a screen for extragenic suppressors of the histidine auxotrophy caused by the his4-917 transposon insertion mutation upstream of the yeast HIS4 gene (31) . The deduced amino acid sequence of the Spt14 protein (19) shows 44% identity to the product of the PIG-A gene, the latter having been cloned by complementation of a GlcNAc-PI synthesis-deficient human cell line (20) . The temperature-sensitive growth phenotype of gpi3 was rescued upon transformation of the cells with centromeric or multicopy plasmids containing the SPT14 gene, and plasmids from a YCp50 genomic DNA library that complemented the temperature sensitivity of gpi3 contained, as judged by restriction endonuclease digestion, the SPT14 gene. Mutations in the SPT14 and PIG-A genes thus, respectively, give rise to GPI anchoring and GlcNAc-PI synthetic defects in yeast and mammalian cells.

Cloning of the GPI2 Gene

The wild type counterpart of the defective gene in the gpi2 mutant was cloned by complementation of that mutant's temperature-sensitive growth phenotype with a yeast genomic DNA library in the centromeric vector YCp50 (24) . Plasmids restoring growth of gpi2 cells at 37 °C were recovered from primary transformants, amplified in Escherichia coli, then retested for their ability to rescue the temperature sensitivity of the gpi2 cells. Five plasmids that complemented the gpi2 mutation were recovered. Restriction endonuclease digestions of the genomic DNA inserts in the complementing plasmids revealed that all five contained common restriction fragments. A 3.8-kilobase HindIII fragment was isolated from plasmid pGC2-8.5 (Fig. 5A) and subcloned into a 2 micron vector, yielding plasmid pSLG21; this plasmid also corrected the temperature sensitivity of the gpi2 mutant.


Figure 5: Cloning of the GPI2 gene. A, subcloning of the yeast genomic DNA insert that complements the gpi2 mutation and sequencing strategy (ND*, not determined). B, DNA and deduced amino acid sequence of the GPI2 gene. C, hydropathy analysis of the Gpi2 protein, performed according to Kyte and Doolittle (33), using the DNA Strider 1.1 sequence analysis program (28). Kb, kilobase.



gpi2 cells harboring pSLG21 were tested for correction of their GPI anchoring defect. Pulse labeling of proteins with [H]inositol at 25 and 38 °C showed that [H]inositol labeling of protein, hence GPI anchoring, is restored in the transformed gpi2 cells (Fig. 4A, lanes5 and 6). Further, in vitro assay of membranes of gpi2 cells harboring pSLG21 showed that GlcNAc-PI synthetic activity is also restored (Fig. 4B, lane4). Introduction of the complementing DNA on a multicopy plasmid did not, however, lead to overexpression of GlcNAc-PI synthetic activity relative to the activity in control, wild type membranes.


Figure 4: Correction of the GPI anchoring defect of the gpi2 mutant by the cloned GPI2 gene. A, restoration of [H]inositol labeling of proteins in transformants. gpi2 cells harboring plasmid pSLG21 (g2 + pSLG21, lanes5 and 6) were pulse labeled with [H]inositol at 25 and 38 °C, and [H]inositol-labeled proteins were separated by SDS-polyacrylamide gel electrophoresis, as described in the legend to Fig. 1. Cultures of wild type cells (WT, lanes1 and 2) and of the gpi2 mutant harboring the vector alone (g2 + v, lanes3 and 4) were radiolabeled in parallel as controls. B, restoration of in vitro GlcNAc-PI synthetic activity in transformants. Mixed membranes prepared from gpi2 cells harboring pSLG21 (g2 + pSLG21, lane4) and from the wild type (lane2) and vector control (lane3) strains used in the experiment in A were assayed for the synthesis of [C]GlcNAc-PI, [C]GlcN-PI, and [C]GlcN-(acylinositol)PI ((acyl-Ins)PI). Incubations were for 10 min. Assay conditions and procedures for isolation, separation, and detection of C-labeled lipids were as detailed in Refs. 18 and 22. A sample of lipids from wild type cells labeled in vivo with [H]inositol was run in lane1 of the chromatogram.



To establish that the cloned gene is indeed the wild type counterpart of the gene that is defective in the gpi2 mutant, the wild type chromosomal sequence that is homologous to the DNA in the plasmid insert was marked with the URA3 gene. To do this, the integrating plasmid pSLG27, which contains the putative GPI2 sequence and the URA3 gene, was linearized by digestion with SalI, which cleaves at a single site in GPI2. The linearized plasmid was introduced into the wild type haploid strain XM37-10C, yielding strains prototrophic for uracil on account of the integration of the URA3 gene at the chromosomal locus homologous to the plasmid's gpi2-complementing insert. Such a haploid was then crossed to a gpi2 ura3 haploid, the resulting diploids induced to sporulate, and the asci submitted to tetrad analysis. All 20 tetrads analyzed yielded two temperature-resistant, Ura segregants and two temperature-sensitive, Ura segregants. The putative GPI2 gene and the integrated URA3 gene are therefore very tightly linked, a finding that indicates that the cloned gene is, indeed, GPI2.

Sequence Analysis of the GPI2 Gene

Subcloning and testing of fragments of the genomic DNA insert containing the GPI2 gene for complementation of the temperature sensitivity of the gpi2 mutant led to the identification of a SalI site likely to lie in the GPI2 coding region (Fig. 5A). DNA sequence diverging from this SalI site was obtained from plasmids pSLG25 and pSLG26 (Fig. 5B). The sequence surrounding the SalI site was verified by sequencing across the SalI junction using primers complementary to GPI2 sequences in pSLG25 and pSLG26. An open reading frame was identified that encodes a predicted protein of 269 amino acids with a molecular weight of 29,590 (Fig. 5C). Initial sequencing of 234 nucleotides of the insert in pSLG21 revealed the presence of another open reading frame whose nucleotide sequence was virtually identical to that of a portion of the S. cerevisiae ATP4 gene, which encodes the ATP synthase chain (32) (Fig. 5A).

Hydropathy analysis of the predicted Gpi2 protein according to Kyte and Doolittle (33) reveals the presence of six stretches of hydrophobic amino acid residues, of which at least two have the width to span the membrane. The presence of potential membrane-spanning regions in the predicted Gpi2 protein is consistent with the fact that Gpi2p-dependent GlcNAc-PI synthetic activity sediments in the yeast-mixed membrane fraction. The Gpi2 protein has no apparent site for cleavage by signal peptidase but contains three Asn-X-Ser/Thr sites that could, potentially, be N-glycosylated.

Comparisions of the Gpi2 protein sequence with those of other proteins in the data base using the BLASTP program (34) showed that the predicted protein sequence of Gpi2 has no similarity to any other protein in the data base, including the known mammalian GPI anchoring proteins PIG-A and PIG-H, the latter being the product of a gene that complements the GlcNAc-PI-synthesis-defective Thy-1 class H lymphoma mutant (35) . In addition, Gpi2p is not similar to Gpi1p.()

Lethal Disruption of the GPI2 Gene

The GPI2 gene was disrupted using the transformation technique (29) (Fig. 6A), as detailed under ``Experimental Procedures.'' Integration of the disrupted gpi2::URA3 gene at the chromosomal GPI2 locus was confirmed by Southern blot analysis (Fig. 6B). Heterozygous gpi2::URA3/GPI2 diploids were induced to sporulate, and the four haploid ascospores in the resulting asci were dissected out and incubated at 25 °C on non-selective medium. In each of the 40 tetrads analyzed, two spores grew into colonies, and two failed to do so. The two viable haploid segregants were always uracil auxotrophs; therefore, the segregants that did not grow harbored the disrupted gpi2::URA3 allele. Microscopic examination of the segregants that failed to give rise to colonies revealed that the ascospores had germinated but subsequently completed no more than four cell divisions. We conclude that the GPI2 gene is essential for vegetative growth of S. cerevisiae.

Overexpression of GPI Genes in Different gpi Mutant Backgrounds

To test whether each GPI gene product is required independently for GPI synthesis, we introduced the cloned GPI2 and SPT14 genes into each of the gpi1, gpi2, and gpi3 mutants on multicopy (2 micron) plasmids (Fig. 7). Overexpression of the SPT14 gene rescues only the gpi3 and spt14 strains; therefore, the SPT14 gene product cannot substitute for the Gpi1 or Gpi2 proteins. Overexpression of GPI2 allows vigorous growth of the gpi2 mutant at 37 °C but not of the gpi3 mutant; the Gpi2 protein therefore does not substitute for the Spt14/Gpi3 protein. However, overexpression of the GPI2 gene does allow slow growth of the gpi1 mutant at 37 °C. Given that the gpi1 and gpi2 mutants have the same biochemical phenotype, an interpretation of this partial multicopy suppression (36) is that the Gpi1 and Gpi2 proteins interact with one another in vivo and that a weak interaction between the two proteins in the gpi1 mutant can be compensated for by overexpression of the Gpi2 partner.


Figure 7: Expression on 2 micron plasmids of the GPI2 and SPT14 genes in the gpi1, gpi2, and gpi3 mutants. Multicopy (2 micron) plasmids containing the GPI2 (pSLG21) or SPT14 (pGY10) genes were introduced into the gpi1, gpi2, gpi3, and spt14-3 mutants. Transformants were tested for growth at 25 and 37 °C on synthetic complete medium (22), plates being incubated for 4 days. As controls, mutants harboring the vectors alone were tested in parallel.



To address the possibility that one or more of the GPI gene products regulates the expression of genes involved in GPI synthesis, we tested whether any of the gpi mutations affects expression of any of the other GPI genes. The results of Northern mRNA hybridization experiments showed that expression of each GPI gene was normal in each of the other gpi mutant backgrounds (not shown). Taken together, our results suggest that the products of the GPI1, GPI2, and SPT14 genes participate directly in the first step in GPI synthesis.


DISCUSSION

Our strategy of screening yeast strains for defects in the incorporation of [H]inositol into protein has led to the identification of two further GPI anchoring mutants, gpi2 and gpi3, whose enzymatic defect is in GlcNAc-PI synthesis, the first step in GPI assembly. The gpi3 mutant is defective in the yeast homolog of PIG-A, the gene that complements a GlcNAc-PI synthesis-defective mutant human cell line (19, 20) . This indicates that the gene products involved in GPI synthesis are likely to be conserved among eukaryotes. The gpi2 mutant is defective in a gene, GPI2, which encodes a protein that shows no similarity to any of the yeast or mammalian proteins known so far to be required for GlcNAc-PI synthesis. Among the mammalian GPI anchoring mutants (7, 8, 9, 10, 11, 12, 13) are three complementation groups whose defect is in GlcNAc-PI synthesis (14, 16, 17) . Our finding that mutations in different yeast genes affect GlcNAc-PI synthetic activity indicates that the requirement of multiple proteins for the first step in GPI synthesis to occur is not confined to mammalian cells but is a general characteristic of GPI synthesis.

Since we have so far isolated single alleles of each of our three GlcNAc-PI synthesis mutants, our screen for mutants blocked in [H]inositol incorporation into protein has not been saturated, and further GlcNAc-PI synthesis mutants may well be found. Indeed, none of the three yeast proteins involved in GlcNAc-PI synthesis resembles the mammalian GPI-H protein (35) , and, since the wild type counterpart of the defective gene in the Thy-1 class C mutant has not been cloned, we do not know whether it will be the counterpart of the GPI1 or GPI2 gene product. Therefore, at least four proteins may be required for GlcNAc-PI synthesis.

The facts that mutations in three genes affect the first step in GPI synthesis in vitro and that none of the gpi mutations affects transcription of the other two GPI genes are most simply interpreted as indicating that these gene products are part of a protein complex. Thus, either GlcNAc-PI synthesis itself requires at least three proteins or, alternatively, a number of the steps in the assembly of the GPI precursor glycolipid may be carried out by an enzyme complex (2) whose in vitro activity is abolished by mutation of one of its subunits. The finding that double gpi mutants are not viable suggests that mutations in two of its members render the putative protein complex non-functional in vivo. The notion that two or more of the Gpi proteins form part of a complex is supported by the finding that overexpression of the GPI2 gene partially suppresses the temperature sensitivity of the gpi1 mutant.

Although our genetic evidence suggests that the Gpi1, Gpi2, and Gpi3 proteins may form part of a complex, mixing experiments with membranes from pairs of gpi mutants did not lead to reconstitution of an active GlcNAc-PI-synthesizing complex, as was the case when lysates of the Thy-1 class A, C, and H lymphoma mutants were mixed (16) . However, it may not be possible in such experiments to effect reciprocally the replacement of an intact but mutant protein from one complex with the non-mutant protein from another protein complex. In addition, membranes from gpi1 cells harboring the GPI2 gene on a multicopy plasmid did not show detectable restoration of GlcNAc-PI synthetic activity.

Our findings that single gpi1, gpi2, and gpi3 mutants are all temperature sensitive for growth, that the GPI2 gene is essential for vegetative growth (as is SPT14/GPI3(19) ), and that double gpi mutants are not viable provide strong support for our notion that GPI synthesis is required for the growth of S. cerevisiae(18) . Cessation of GPI synthesis may contribute to the phenomenon of ``inositol-less death,'' which ensues upon inositol starvation of inositol auxotrophs and which may be the result of a block in cell surface expansion (reviewed in Ref. 37). Indeed, abolition of GPI synthesis may interfere with cell wall assembly by preventing the proposed transfer of wall glycoproteins from a GPI-anchored intermediate to an acceptor glucan in the wall (6) . Intracellular events are also influenced in cells with a GPI anchoring deficiency. Thus, the gpi3/spt14 mutations, as well as the gpi1 and gpi2 mutations (not shown), suppress the his4-917 transposon insertion mutation. One possibility is that a reduced ability of the mutants to attach GPI anchors to certain cell surface proteins, even at the cells' permissive growth temperature, might mimic a signal that, for example, ultimately halts Ty element transcription.

Our analyses of the gpi mutants we have isolated by screening for defects in [H]inositol incorporation into protein are revealing the complexity of GPI biosynthesis and highlighting the importance of GPIs in a unicellular eukaryote; furthermore, these temperature-sensitive gpi mutants are allowing us to isolate genes for novel proteins involved in GPI anchoring.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM46220 (to P. O.) and GM40306 (to J. F.), by a Junior Faculty Research Award from the American Cancer Society (to P. O.), and by an award from the Central Investment Fund for Research Enhancement of the University of Iowa (to J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U23788.

§
To whom correspondence should be addressed: Dept. of Biochemistry, 309 Roger Adams Laboratory, University of Illinois at Urbana-Champaign, 600 South Mathews Ave., Urbana, IL 61801. Tel.: 217-333-4139; Fax: 217-244-5858.

The abbreviations used are: GPI, glycosyl phosphatidylinositol; PI, phosphatidylinositol; GlcN-PI, glucosaminyl phosphatidylinositol; Dol-P, dolichyl phosphate; Dol-PP, dolichyl pyrophosphate.

S. D. Leidich and P. Orlean, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Sunil Lal and John Abelson for making the bank of temperature-sensitive mutants available, and we are grateful to Dr. Mark Goebl for carrying out protein sequence data base searches. We also thank Dr. Anthony Crofts for advice on analyses of hydropathy.


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