From the
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 [
Diverse proteins in eukaryotic cells are anchored in the
external face of the plasma membrane by a GPI
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 [
Growth media and materials were as described in
Ref. 18. Synthetic complete medium was prepared as described in Ref.
22. Screen for Defects in [
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
Mutants 27-5A and
53-12C showed a temperature-sensitive defect in
[
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).
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.
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.
Our strategy of screening yeast strains for defects in the
incorporation of [
Since we have so
far isolated single alleles of each of our three GlcNAc-PI synthesis
mutants, our screen for mutants blocked in
[
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
[
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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) .
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.
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.
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/NH
OH
(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.
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).
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).
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/NH
OH (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.
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.
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).
(
)
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.
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.
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.
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.
/EMBL Data Bank with accession number(s) U23788.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.