From the Institute of Biochemistry, University of Fribourg, Chemin du Musée 5, CH-1700 Fribourg, Switzerland
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ABSTRACT |
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Gpi7 was isolated by screening for
mutants defective in the surface expression of
glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants
are deficient in YJL062w, herein named GPI7. GPI7 is not
essential, but its deletion renders cells hypersensitive to Calcofluor
White, indicating cell wall fragility. Several aspects of GPI
biosynthesis are disturbed in Glycosylphosphatidylinositol
(GPI)1-anchored proteins
represent a subclass of surface proteins found in virtually all
eukaryotic organisms (1). The genome of Saccharomyces
cerevisiae contains more than 70 open reading frames (ORFs)
encoding for proteins that, as judged from the deduced primary
sequence, can be predicted to be modified by the attachment of a GPI
anchor (2, 3). In about 25 of them, the presence of an anchor has been
confirmed biochemically. A majority of them lose part of the anchor and become covalently attached to the For the biosynthesis of GPI anchors, phosphatidylinositol (PI) is
modified by the stepwise addition of sugars and ethanolamine phosphate
(EtN-P), thus forming a complete precursor lipid (CP) which
subsequently is transferred en bloc by a transamidase onto newly synthesized proteins in the ER (7, 8). The identification of
genes involved in the biosynthesis of the CP and its subsequent attachment to proteins has been possible through the complementation of
mammalian and yeast gpi Here we report on the characterization of gpi7. Four
independent gpi7 mutants accumulated M4, an abnormal GPI
intermediate that is less hydrophilic than CP2, the precursor
accumulating when the transfer of GPIs to proteins is interrupted (18,
19, 21, 23). Our preliminary characterization of M4 had shown that
deacylation by NH3 followed by HF treatment, used to
hydrolyze selectively the phosphodiester bonds (Fig.
1), yielded the same Man4-GlcN-inositol fragment as CP2, and we speculated that
gpi7 mutants may be unable to add the EtN-P onto Man3 (Fig.
1) (21). Here we show that this speculation was wrong, that CP2 differs from M4 with regard to a previously unrecognized side chain attached to
Man2 (Fig. 1), and that GPI7 is required for the attachment of this side chain.
Strains, Growth Conditions, and Materials--
S.
cerevisiae strains were FBY11 (MATa ade2-1
ura3-1 leu2-3,112 trp1-1 his3-11,15 gpi8-1), FBY15 (MAT
Materials were obtained from the sources described recently (22).
Cysteamine was from Sigma; [3H]dihydrosphingosine was
synthesized as described (24); myriocin was a kind gift of Dr. N. Rao
Movva (Novartis, Basel, Switzerland); antibodies to Och1p, alkaline
phosphatase, Kex2p, and Wbp1p were kindly donated by Dr. Y. Jigami,
National Institute of Bioscience and Human Technology, Ibaraki 305, Japan; Dr. S. Emr, Howard Hughes Medical Institute, University of
California, San Diego; Dr. R. Fuller, University of Michigan Medical
Center, Ann Arbor, MI; and Dr. M. Aebi, Mikrobiologisches Institut, ETH
Zürich, Switzerland, respectively.
Cloning, Partial Sequencing, and Disruption of GPI7--
The
GPI7 gene was cloned by complementation of the ts growth
phenotype of the gpi7-1/gpi8-1 double mutant as described
(19). The three plasmids complementing gpi7-1 contained a
4.3-kb common DNA restriction fragment that was partially sequenced by
the dideoxy sequencing method (25). The complementing insert
SphI/SspI of 3.6 kb was cloned into the
SphI/SmaI-digested YEp352 multicopy vector (26)
or YCplac33 single copy vector (27) to generate pBF41 (Fig.
3C) and pBF43, respectively.
One step disruption of GPI7 was done as described (28).
Briefly, the 1.5-kb long KanMX4 module was PCR-amplified by using pFA6a-KanMX4 as template and the following two adapter primers: GPI7-forwards
(5'-CTTCACCAAGTTAGCAAGATGAACTTGAAGCAGTTCACGTGCCtcgatgaattcgagctc-3') with 17 nucleotides (nt) of homology to the pFA6a-KanMX4 multiple cloning site (in lowercase) and 43 nt of homology to
GPI7 (in uppercase) starting 18 nt upstream of the start
codon (bold); GPI7-backwards
(5'-ATCAAGAGCGCAAAGGAGGGCCAATTCAGGTAACCAGCCATTCAcgtacgctgcaggtcgac-3') with 18 nt of homology to the pFA6a-KanMX4 multiple cloning site (lowercase) and 44 nt of homology to the ORF of
GPI7 in the region immediately upstream of the stop
codon. This PCR DNA fragment was used to transform the diploid strain
FBY118, homozygous for GPI7, and FBY40, a heterozygous
gpi7-1/GPI7 strain. The correct targeting of the PCR-made
KanMX4 module into the GPI7 locus in geneticin-resistant
clones was verified by PCR with whole yeast cells using primers
GPI7-plus (5'-GTTCATCTACCACGCAC-3') starting 36 nt upstream of the ATG,
GPI7-minus (5'-GACCCAAGTAATGCAGG-3') starting 631 base pairs downstream
of the ATG and the K2 primer of the KanMX4 module
(5'-GTATTGATGTTGGACG-3').
Purification of Recombinant His-tagged Gpi7p and Antibody
Production--
Plasmid pBF41 (Fig. 3C) was digested with
BstYI and EcoRV to generate a 633-base pair
fragment of GPI7. This fragment was inserted into the
multiple cloning site of the bacterial expression vector pQE-30
(Qiagen) digested with BamHI/SmaI thus generating the plasmid pBF402. This plasmid was used to transform the E. coli strain M15[pREP4]. Expression of the recombinant protein was induced with isopropyl-1-thio- Membrane Association, Protease Sensitivity, and Cellular
Localization of Gpi7p--
The nature of the association of Gpi7p with
the membrane was determined following a previously described protocol
(30) except that the EDTA concentration in buffer G was increased from
2 to 20 mM. Gpi7p protease sensitivity was examined by
proteinase K digestion of microsomes essentially as described (31).
Briefly, 100 A600 of washed W303 cells were
resuspended in 1 ml of lysis buffer (20 mM HEPES, pH 7.5, 500 mM sucrose, 3 mM magnesium acetate, 20 mM EDTA, 1 mM dithiothreitol) and were lysed by
agitation with glass beads at 4 °C. The homogenate was centrifuged
for 5 min at 600 × g to remove unbroken cells, and the
supernatant was centrifuged for 15 min at 13,000 × g.
The membrane pellet was resuspended in 240 µl of the same lysis
buffer and split into 6 aliquots of equal size. Aliquots of microsomes
were incubated for 20 min on ice with or without 0.5% Triton X-100 and
proteinase K. Digestions were stopped by addition of
phenylmethylsulfonyl fluoride (final concentration 4 mM,
added from a 200 mM stock in ethanol) and kept on ice for
an additional 10 min before being boiled in sample buffer (32). The
subcellular localization of Gpi7p was determined essentially as
described (33). Briefly, 500 A600 units of
mid-log phase W303-1B cells were broken by agitation with glass beads in 200 mM sorbitol, 25 mM PIPES, pH 6.8, 50 mM KCl, 5 mM NaCl, 10 mM EDTA, 10 mM NaN3/NaF, 1 mM
phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and antipain, each
at 30 µg/ml). After removal of the unbroken cells the homogenate was
centrifuged for 10 min at 8,000 × g at 4 °C to
generate pellet P8 and supernatant S8. S8 was divided in two and either
precipitated by the addition of trichloroacetic acid to 10% or
centrifuged at 100,000 × g for 1 h to generate
pellet P100. For zymolyase treatment the cells were washed and
resuspended at 50 A600 units/ml in zymolyase
buffer (1.2 M sorbitol, 50 mM
K2HPO4 pH 7.5, 40 mM
2-mercaptoethanol, 20 mM EDTA, 10 mM
NaN3, 10 mM NaF) containing zymolyase 20T.
After a 40-min incubation at 30 °C, cells were placed on a cushion
of 1.5 M sorbitol, 50 mM
K2HPO4, pH 7.5, 20 mM EDTA, 10 mM NaN3, 10 mM NaF and were
centrifuged. For Western blotting, all the samples were denatured
during 5 min at 95 °C in reducing sample buffer and run on a 6, 7.5, or 10% SDS-PAGE for detection of antigens, respectively (32). Western
blotting was carried out with anti-Wbp1p, anti-alkaline phosphatase,
anti-Gas1p, or anti-Kex2p antisera or with affinity purified anti-Gpi7p
or anti-Och1p antibodies, always using the chemiluminescence ECL kit
from Amersham Pharmacia Biotech, Buckinghamshire, UK.
Labeling of Cells--
Previously described procedures were used
to label cells with [2-3H]Man (23),
[4,5-3H]DHS, or [2-3H]Ins (24) and to label
microsomes with UDP-[3H]GlcNAc (22). Delipidated protein
extracts for SDS-PAGE and lipid extracts were made as described (24).
Lipid extracts were analyzed by ascending TLC using 0.2-mm thick silica
gel 60 plates with the solvent 1 (chloroform/methanol, 0.25% KCl in
water, 55:45:10, v/v) or solvent 2 (chloroform/methanol/water, 10:10:3,
v/v). Radioactivity was detected and quantitated by one- and
two-dimensional radioscanning (LB 2842; Berthold AG, Regensdorf,
Switzerland). TLC plates were sprayed with EN3HANCE and
exposed to film (X-Omat; Eastman Kodak Co.) at Analytical Methods--
Lipid extracts were deacylated with NaOH
(34) and treated with JBAM (35) as described. For GPI-PLD treatment
lipid extracts were dissolved in 20 mM Tris-HCl, pH 7.4, 0.1 mM CaCl2, 20% 1-propanol. Incubations were
for 12 h at 37 °C. All treated lipid extracts were desalted by
partitioning between n-butyl alcohol and an aqueous solution
of 0.1 mM EDTA, 5 mM Tris-HCl, pH 7.5, and back
extraction of the butanol phase with water before TLC (23). Lipids were treated with methanolic NH3 to remove the acyl group on Ins
(36) and cleaved using nitrous acid (37) as described. Lipids were purified by preparative TLC on 0.2-mm thick Silica Gel 60 plates (Merck, Germany) in solvent 2. Radioactive spots were localized by
radioscanning, scraped, and eluted with solvent 2. A second run on TLC
was done to obtain radiochemically pure M0, M4, and CP2.
Soluble head groups were obtained from lipids through GPI-PLD treatment
done as above, followed by limiting methanolic NH3 deacylation (36). Non-hydrolyzed GPIs were removed by butanol extraction (23). The water-soluble head groups were treated with JBAM
(0.5 units) or ASAM (5 microunits) as described (38). HF
dephosphorylation was done as described (39). The generated fragments
were analyzed by paper chromatography in methylethyl ketone/pyridine/H2O (20:12:11) as described (39). Before
paper chromatography the products were N-acetylated and
desalted over mixed-bed ion exchange resin AG-501-X8 (Bio-Rad) unless
indicated otherwise (34). Acetolysis was done as described (40).
Radiolabeled Manx-GlcNAc-[3H]Ins
(x = 1, 2, 3, 4) chromatography standards (Figs. 5 and
6, standards 1-4) were generated through fragmentation of
[3H]Man-labeled head groups of CP2, isolated from
pmi40 by acetolysis then HF, HF then ASAM, JBAM then HF, and
HF treatments, respectively. The GlcNAc-[3H]Ins standard
(Figs. 5 and 6, standard 0) was generated by HF treatment of
the [3H]Ins-labeled head group of M0, obtained from
sec53 cells. All standards were N-acetylated.
Dionex HPLC analysis of non-dephosphorylated head groups was done
exactly as described (23). Anchor peptides were prepared from labeled
proteins as described (23).
Limiting HF Treatment of Head Groups--
For limiting HF
treatment, aliquots of radiolabeled head groups derived from CP2 and M4
and prepared as above were dephosphorylated with 50 µl of 48%
aqueous HF at 0 °C as described (38) for 0-28 h. After
neutralization with saturated LiOH, samples were desalted by gel
filtration through an 8-ml Sephadex G-10 (Amersham Pharmacia Biotech)
column. Samples were then dried in the Speed-Vac and treated with JBAM
prior to complete HF dephosphorylation (60 h, 0 °C). Samples were
neutralized again with LiOH and N-acetylated. Aliquots were
dried and then directly applied to Whatman paper No. 1M and analyzed by
descending chromatography as described above.
Cloning of GPI7--
As reported
before (19) and shown in Fig.
2, wild type (wt) cells do not contain
polar GPIs (lane 1), gpi8-1 accumulates CP2 as
the most polar GPI lipid (lane 8), and gpi7-1 and
the gpi7-1/gpi8-1 double mutant accumulate M4 (lanes
4 and 6), thus demonstrating that gpi7-1 is
epistatic to gpi8-1 and suggesting that, during GPI
biosynthesis, Gpi7p may act before Gpi8p. Although the original gpi7 mutants and the unrelated gpi8-1 mutant were
not significantly temperature-sensitive (ts) for growth, the growth of
the gpi7-1/gpi8-1 double mutant was strongly
temperature-dependent. Transfection of a genomic library
into this double mutant allowed the isolation of clones containing
complementing plasmids (19). These clones were labeled with
myo-[3H]inositol ([3H]Ins) at
37 °C, and the lipids were extracted and analyzed by TLC. Upon
transfection some gpi7-1/gpi8-1 indeed had regained the
ability to make CP2 (Fig. 2, lanes 6 and 7) and
showed the same lipid profile as gpi8-1 (Fig. 2, lane
8). All these clones harbored plasmids containing YJL062w as the
only complete ORF. Transfection of a multicopy vector containing
YJL062w under its own promoter (pBF41, Fig.
3C) into gpi7-1
almost completely cured the accumulation of M4 (Fig. 2, lane
5). As expected, the accumulation of CP2 by gpi8-1 was
not abolished by the overexpression of YJL062w (Fig. 2, lanes
8 and 9). YJL062w predicts an 830-amino acid membrane protein with an N-terminal signal sequence for insertion into the ER, 5 potential N-glycosylation sites, and about 9-11 putative transmembrane domains (Fig. 3, A and B). YJL062w
was deleted and replaced by the selectable marker KanMX4. On rich
medium the deletants grew about as rapidly as wt cells at all
temperatures. Thus, YJL062w is not an essential gene. We were unable to
sporulate Characterization of the GPI Intermediate M4--
We found that M4,
contrary to our initial expectation, contained an HF-sensitive group on
Man3 (Fig. 1). Indeed, treatment of the lipid extracts of
gpi7-1 with jack bean
Having recently discovered an additional EtN-P on Man1 of CP2 (22), we
considered the possibility that M4 may be lacking EtN-P on Man1. We
thus proceeded to compare the non-dephosphorylated head groups of M4
and CP2 by Dionex HPLC using a system in which the presence of
negatively charged phosphodiesters greatly retards the elution of
oligosaccharides (42). The non-dephosphorylated head groups of M4 and
CP2 eluted as sharp peaks at fractions 22 and 31, respectively (not
shown). This wide separation suggested that the head group of M4
contains less negative charge than the one of CP2. To assay directly
for a side chain on Man1 of M4, the head group of
[3H]Ins-labeled M4 was first cleaved by acetolysis, a
procedure which, under mild conditions, selectively cleaves
The Lipid Moieties of GPI Intermediates in Lack of Gpi7p Affects the in Vitro Biosynthesis of GPI Precursor
Lipids--
When yeast microsomes are incubated in the presence of
UDP-[3H]GlcNAc, ATP, coenzyme A, GDP-Man, and
tunicamycin, they generate labeled GPI intermediates as the only kind
of labeled lipids (22, 47). Wild type microsomes make GPI intermediates
up to CP2. Although a large array of incomplete intermediates is also
generated, the pattern of labeled intermediates is fairly reproducible.
When we used Characterization of Gpi7p--
Gpi7p was characterized using
affinity purified rabbit antibody made against the N-terminal,
hydrophilic part of GPI7 (Fig. 3B). As shown in
Fig. 8A, the antibody
recognized a heterogeneously glycosylated 208-kDa protein, the
estimated molecular mass of various glycoforms ranging, after heavy
exposure, from about 130 to 230 kDa (Fig. 8A, lane 2). The
predicted mass of the protein before and after removal of the signal
sequence is 94,832 and 92,207 Da, respectively. In glycosylation
mutants
The cellular localization of Gpi7p was investigated by subcellular
fractionation as shown in Fig. 8D. Differential
centrifugation at 8,000 and 100,000 × g for 10 and 60 min, respectively, achieved satisfactory separation of the Golgi
markers Kex2p and Och1p from the ER marker Wbp1p and from the vacuolar
alkaline phosphatase. We were concerned that the relative amounts of
these proteins in the 100,000 × g pellet may be
underestimated due to ongoing proteolytic degradation during the
100,000 × g spin. Therefore the supernatant of the
8,000 × g spin was split whereby proteins were
immediately precipitated with trichloroacetic acid in one half, and the
other half was pelleted at 100,000 × g. Gpi7p was exclusively found in the 8,000 × g pellet (P8) and
thus is associated with either the ER, the vacuole, or the PM but not
with the Golgi. As shown in Fig.
9A low amounts of proteinase K
added to intact cells hydrolyzed all of the mature Gpi7p. In contrast,
Wbp1p and the 105-kDa ER form of Gas1p were completely resistant to
this treatment unless membranes were permeabilized with Triton X-100 (Fig. 9A, lane 6). The mature 125-kDa form of Gas1p was
found to be partially resistant to proteinase K, indicating either a tighter interaction of Gas1p with some cell wall components or the
existence of an internal pool of Gas1p as proposed earlier (50). Crude
zymolyase treatment of intact cells also removed all of the mature form
of Gpi7p (Fig. 9B), whereas recombinant zymolyase left Gpi7p
intact (not shown). Longer exposures showed the presence of several
minor bands of smaller size all of which were also present in
Deletion of GPI7 Alters GPI Protein Transport and
Remodeling--
We previously reported on the accumulation of the
immature 105-kDa ER form of Gas1p in gpi7 mutants (21). We
therefore investigated GPI protein transport in
The lipid remodeling of GPI anchors is significantly altered in
Yeast and mammals contain the same GPI carbohydrate core
structure. This suggests that the GPI anchoring pathway has been established early in evolution and has rigorously been conserved in
widely diverging organisms. On the other hand, the side chains added to
this core as well as the lipid moieties of the anchor tend to vary a
lot between different species (1). The GPI anchors of S. cerevisiae contain two types of side chains as follows: one or two
mannoses are linked to Man3 (53) and an EtN-P side chain is linked to
Man1.3 Both side
chains are already present on the precursor lipid CP2 (22, 23). These
two side chains are also found in some vertebrates, including mammals,
and possibly in Dictyostelium discoideum (1), suggesting
that not only the GPI core structure but also certain kinds of side
chains have been invented in and conserved since early times of
evolution. Here we present evidence for yet a further, possibly
conserved HF-sensitive substituent on CP2 which is attached to Man2. So
far, the only side chain attached to Man2 reported in the literature is
EtN-P. EtN-P was found by mass spectrometry on 15% of anchors of human
erythrocyte cholinesterase and 3% of bovine liver 5'-nucleotidase (44,
46). Partial acid hydrolysis has also indicated an HF-sensitive
substituent on Man2 in 40% of CD52-II (45). Analysis of the
ethanolamine/Ins ratio in GPI anchors of porcine renal membrane
dipeptidase and of human placental alkaline phosphatase yielded values
of 2.5 and 2.4, suggesting the presence of EtN-P on Man2 in 50 and 40%
of their anchors, respectively (54, 55). However, no such side chain
was detected in other mammalian GPI proteins such as rat brain Thy-1
glycoprotein (56) or hamster scrapie prion (57). It may be that in many studies part or all of EtN-P side chains on Man2 and Man1 were hydrolyzed by an unspecific phosphodiesterase during the purification of the respective GPI proteins and preparation of anchor peptides using
Pronase. Such a phosphodiesterase activity may explain why we failed to
detect any EtN-P side chain on GPI-anchored yeast proteins in the past
(53), although we now have firm evidence for the presence of an
HF-sensitive substituent on Man1.3 The chemical nature of
the side chain on Man2 of CP2 remains to be determined. The presence of
an HF-sensitive side chain on Man2 of CP2 has its parallel in human
cells. Indeed, there is evidence for an HF-sensitive group on Man2 of
H8, the most polar GPI lipid of HeLa cells (58). The EtN-P side chains
on Man1 and Man3 being conserved between mammalian organisms and yeast, it appears reasonable to speculate at this point that the analogy between mammalian and yeast anchors may extend to the substituent on
Man2, i.e. that also the side chain on Man2 of yeast GPI
structures may consist of an EtN-P and that this EtN-P may be present
on some mature GPI proteins of S. cerevisiae.
Our data further show that ceramide remodeling in the Golgi/PM is
significantly reduced in Our previous data suggested that CP2 can be transferred to proteins
(23), and our working hypothesis until recently assumed that CP2
represents the GPI lipid used for GPI anchoring also by normal cells
that do not accumulate this lipid ("CP2 hypothesis"). By
consequence we would have predicted that all the enzymes required for
the elaboration of CP2 are localized in the ER. Paradoxically, the
subcellular fractionation experiments and protease treatment of intact
spheroplasts strongly suggest that the bulk of Gpi7p resides at the
cell surface (Fig. 9, A and B). Moreover,
although we recently succeeded in demonstrating the presence of an
HF-sensitive group on Man1 of immature ER forms of GPI proteins, we
presently lack the tools to look for such a group on Man2. Thus, the so far available data raise a doubt whether it is CP2 which is added to
GPI proteins in the ER, and we therefore are presently considering the
possibility that other GPI lipids than CP2 are the physiological substrate of the ER transamidase. In fact, neither CP2 nor M4 can be
detected in wt cells. It therefore seems possible that under
physiological conditions cells add M4 to GPI proteins ("M4 hypothesis") and that CP2 is elaborated only in mutants in which M4
cannot be transferred to proteins, spills out of the ER, and reaches
the PM. It is noteworthy that Homology searches show that two ORFs of S. cerevisiae are
related to GPI7, MCD4 (= YKL165c), and YLL031c.
They belong to a novel gene family comprising for the moment the nine
members shown in Table II which, based on
the many predicted transmembrane domains, were previously classified as
putative permeases (60). Pairwise alignment allows us to group them
into three subfamilies of more closely related ORFs. All nine ORFs
predict membrane proteins of about 100 kDa having an N-terminal signal
sequence, a hydrophilic N-terminal part, and multiple transmembrane
domains in their C-terminal half. mcd4 mutants were obtained
in a screen for cells deficient in the cell cycle controlled
polarization of growth, a phenotype also generated by mutations in the
exocyst or in N-glycosylation (61). The subfamilies typified
by GPI7 and YLL031c are more closely related to each other
than to the MCD4 subfamily. All nine family members contain
two conserved motifs at about the same position in the hydrophilic
N-terminal domain, namely HXLGXDXXGH and
DHGMXXXGXHG. These motifs are also found in two
EST clones from human cDNA that have high homology to
MCD4 (NCBI PID 1779747 and 1765215, ClustalW alignments
giving aligned scores of 46 and 35). Very interestingly, by a
reiterated Psi Blast search at the National Center of Biotechnology
Information (NCBI) (62) one can find a highly significant homology of
all three subfamilies with a large family of phosphodiesterases. The
large majority of these homologous sequences encode mammalian
cell-surface proteins classified as alkaline phosphodiesterase I,
nucleotide pyrophosphatase, or alkaline phosphatase. The homology
comprises a region of about 220-240 amino acids in the N-terminal
hydrophilic part of GPI7, YLL031c, and MDC4. The
homology of GPI7 in this region with mammalian and plant
phosphodiesterases amounts to 17-18% identity and 30-34% similarity
and comprises a motif
PTXTX8TGX2P
which is common to bacterial, viral, plant, and mammalian
phosphodiesterases. This homology may suggest that Gpi7p itself is the
transferase adding the phosphodiester-linked substituent on Man2. In
this context it is interesting to note that the EtN-P on Man3 of the
GPI anchor has been shown to be transferred by transesterification
using phosphatidylethanolamine as donor of EtN-P (63, 64). Mutants in
YLL031c also accumulate abnormal GPI intermediates which on TLC have
about the same mobility as M4 suggesting that YLL031c is
similarly involved in adding
EtN-P.2 Thus, it
is conceivable that not only the GPI7 subfamily but also
other subfamilies are involved in the transfer of EtN-P onto the GPI
core structure. However, the transesterification activity of Gpi7p will
have to be shown directly before one can exclude that the primary
function of Gpi7p is to generate some signal from the cell wall which
regulates GPI protein transport and remodeling as well as side chain
addition to GPI structures.
gpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of
GPI anchors by ceramide, is significantly reduced, and the transport of
GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9-11 predicted transmembrane domains in
the C-terminal part and a large, hydrophilic N-terminal ectodomain. The
bulk of Gpi7p is located at the plasma membrane, but a small amount is
found in the endoplasmic reticulum. GPI7 has homologues in
Saccharomyces cerevisiae, Caenorhabditis
elegans, and man, but the precise biochemical function of this
protein family is unknown. Based on the analysis of M4, an abnormal GPI
lipid accumulating in gpi7, we propose that Gpi7p adds a
side chain onto the GPI core structure. Indeed, when compared with
complete GPI lipids, M4 lacks a previously unrecognized
phosphodiester-linked side chain, possibly an ethanolamine phosphate.
Gpi7p contains significant homology with phosphodiesterases suggesting
that Gpi7p itself is the transferase adding a side chain to the
1,6-linked mannose of the GPI core structure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,6-glucans of the cell wall (4-6). A minority of GPI proteins retain the GPI anchor in an intact
form and stay at the plasma membrane (PM).
mutants, i.e. mutants
being deficient in GPI anchoring of membrane proteins (7, 9-20). In
our laboratory, a series of recessive gpi
mutants
(gpi4 to gpi10) has been obtained by screening
for yeast mutants that are unable to display the GPI-anchored
-agglutinin (Sag1p) at the outer surface of the cell wall, although
the synthesis and secretion of soluble proteins is normal (21, 22).
View larger version (16K):
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Fig. 1.
Presumed structure of the complete yeast
precursor glycolipid CP2. Relevant cleavage procedures are
indicated. Man1, Man2, Man3, and Man4 designate the 1,4-linked,
1,6-linked, and
1,2-linked mannoses (Man). X indicates
an HF-sensitive group that is not yet defined chemically. R,
alkyl; P, phosphate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ade2-1 ura3-1 leu2-3,112 trp1-1 his3-11,15 gpi7-1), W303-1B
(MAT
ade2-1 can1-100 ura3-1 leu2-3, 112 trp1-1
his3-11,15), X2180-1A (MATa
lys
), FBY122 (MATa ade2-1 ura3-1
leu2-3,112 trp1-1 his3-11,15 gpi8-1 gpi7-1), FBY182 (MAT
ade2-1 ura3-1 leu2-3,112 his3-11,15 gpi7::KanMX4),
HMSF176 (MATa sec18-1), FBY49 (MATa
sec18-1 gpi7::KanMX4), C4 (MATa
ura3-52 leu2-3,112 pmi40), HMSF331 (MATa
sec53-6), LB2134-3B (MATa mnn9), and
YNS3-7A (MATa ura3 his
mnn1
och1::LEU2). Diploid strains were FBY118
(MATa/
ade2-1/ade2-1 ura3-1/ura3-1 leu2-3,
112/leu2-3,112 TRP1/trp1-1 his3-11,15/his3-11,15
LYS/lys
), FBY40 (MATa/
ade2-1/ade2-1
ura3-1/ura3-1 leu2-3,112/leu2-3,112 TRP1/trp1-1 his3-11,15/his3-11,15
LYS/lys
GPI7/gpi7-1), and FBY43
(MATa/
ade2-1/ade2-1 ura3-1/ura3-1 leu2-3,112/leu2-3,112 TRP1/trp1-1 his3-11,15/his3-11,15
LYS/lys
gpi7::KanMX4/gpi7::KanMX4). Maintenance and
growth conditions have been described (19). The absorbance of dilute
cell suspensions was measured in a 1-cm cuvette at 600 nm, and one
A600 unit of cells corresponds to 1-2 × 107 cells depending on the strain. Escherichia
coli strains were HB-101, XL1 blue, and M15 [pREP4] (Qiagen).
-D-galactopyranoside
and purified on a nickel-nitrilotriacetic acid-agarose column (Qiagen)
under denaturing conditions according to the manufacturer's
instructions. A polyclonal antiserum was raised against this Gpi7p
fragment by repeated intramuscular injections of 100 µg of
recombinant protein into a rabbit. Ten mg of the recombinant protein
were coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia
Biotech) according to the manufacturer's instructions, and antiserum
was affinity purified as described (29).
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
YJL062/
YJL062 diploids indicating that YJL062 is
required for sporulation. However,
YJL062/YJL062 heterozygotes
sporulated readily and
YJL062 spores germinated normally. In
accordance with previous results on gpi7 mutants (21),
growth of
YJL062 (=
gpi7, see below) on plates at
37 °C was severely inhibited by 0.5 mg/ml Calcofluor White.
YJL062 accumulated M4 at even higher levels than gpi7-1,
and this accumulation was almost completely suppressed by the
transfection of pBF41 (Fig. 2, lanes 2 and 3).
Residual accumulation of M4 may be due to some cells that lost the
complementing plasmid. Transfection of YJL062w under its own promoter
on a single copy vector (plasmid pBF43) was sufficient to suppress the
accumulation of M4 in a homozygous
YJL062/
YJL062 diploid (Fig. 2,
lanes 11 and 12). As can be seen in Fig. 2,
gpi7-1,
YJL062,
YJL062/
YJL062, and
gpi8-1 mutants also show minor amounts of the
GlcN
1,6(acyl
)Ins-P-DAG GPI intermediate M0, the accumulation of
which is believed to reflect a build up of GPI intermediates throughout
the biosynthetic pathway (Fig. 2, lanes 2, 4, 6, 8, and
11). (It should be noted that some intermediates of
intermediate size are obscured on TLC by PI and inositol
phosphoceramide (41).) As expected, expression of YJL062w abolishes the
accumulation of M0 in gpi7-1 and
YJL062 (Fig. 2,
lanes 3, 5, and 12) but not in
gpi7-1/gpi8-1 nor gpi8-1 (lanes 7 and
9), since in the latter the GPI biosynthesis remains blocked. To evaluate if the mutation in gpi7-1 is
genetically linked to YJL062w, YJL062w was disrupted in a heterozygous
gpi7-1/GPI7 diploid. Correct replacement of one
YJL062w locus was verified by PCR in two independent
geneticin-resistant transformants. The verified deletants were
sporulated, and a total of 26 complete tetrads was labeled with
[3H]Ins to analyze the accumulation of M4. In all 26 tetrads only two of the four segregants showed accumulation of M4,
whereas the other two showed the lipid profile of wt cells. Geneticin resistance also segregated 2:2 and cosegregated with M4 accumulation in
all cases. This demonstrates that the mutation of gpi7-1 is tightly linked to YJL062w which we henceforth call GPI7.
Since a construct containing only 348 nucleotides 5' of the initiation codon of GPI7 still retained significant complementing
activity, we also can dismiss the possibility that the complementing
activity of pBF41 is due to one of the two small ORFs located on the
opposite strand in the 5' upstream region of GPI7 and
starting at
409 and
503 with regard to the start codon of
GPI7.
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Fig. 2.
Complementation of gpi7
mutants by YJL062w. Exponentially growing cells were
radiolabeled at 37 °C with [3H]Ins (2 µCi/A600), and desalted lipid extracts were
analyzed by TLC (solvent 2) and fluorography. The same amount of
radioactivity was spotted in each lane. M(IP)2C,
inositol phosphomannosylinositol phosphoceramide. The upper part of the
fluorogram was scanned at higher sensitivity to bring into view the
faint bands of M0. Lane 10 contains
[3H]Ins. Samples still contain residual amounts of free
[3H]Ins after extraction into butanol. The band migrating
between CP2 and M4 in gpi8-1 is a GPI-PLD-, mild base-, and
JBAM-sensitive GPI intermediate containing the
Man4-GlcN-Ins core,2 but the structural
differences between this species and CP2 or M4 have not been
identified.
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Fig. 3.
Sequence of GPI7. A,
potential N-glycosylation sites are shown in
boldface. The predicted signal peptide is
underlined. B shows a hydrophobicity plot
according to Kyte and Doolittle, in which the hydrophobic sequences get
a positive score. C shows the restriction map of pBF41. The
3.6-kb insert (34-3604) contains GPI7 (=
YJL062w, broad arrow). The EcoRV/BstYI
fragment was expressed in bacteria to get antigen for raising
antibodies.
-mannosidase (JBAM, an
exomannosidase) shifted M4 to a slightly less hydrophilic position on
TLC (Fig. 4A, lanes 1 and
2) but not to the position of M0. It seemed conceivable that
JBAM did not remove more than one Man from M4 because it was sterically
hindered by the detergent micelle in which M4 was embedded. To
circumvent this problem, M4 was purified by preparative TLC, and its
hydrophilic head group was liberated by GPI-PLD,
O-deacylated by NH3, and then subjected to
several treatments as indicated at the top of Fig.
5, A-D. The
N-acetylated fragment comigrated with the
Man4-GlcNAc-Ins standard (Fig. 5A). When treated
with JBAM before HF, the resulting N-acetylated fragment comigrated with the Man3-GlcNAc-Ins standard, clearly
indicating the presence of a blocking group on Man3 (Fig.
5B). The blocking group on Man3 was HF-sensitive, since JBAM
done after HF produced a fragment comigrating with GlcNAc-Ins (Fig.
5C). Aspergillus satoi
-mannosidase (ASAM), a
linkage-specific
1,2-exomannosidase, when used after HF treatment,
produced Man2-GlcNAc-Ins (Fig. 5D). The
migration of the fragments shown in Fig. 5, A and
B, was much slower when N-acetylation was omitted
(not shown). This partial characterization of M4 is consistent with the
presence of a classical Man
1,2(EtN-P
)Man
1,2Man
1,6Man
1,4-GlcN
1,6Ins
core structure.
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Fig. 4.
Characterization of M4. A,
gpi7-1 cells were preincubated at 37 °C for 20 min and
were labeled with [3H]Ins (2 µCi/A600). Lipid extracts were treated (+) or
control incubated ( ) with either JBAM or GPI-PLD or were deacylated
by mild base treatment (NaOH). Desalted products were analyzed by TLC
(solvent 2) and fluorography. B, sec53 and
gpi7 were preincubated and labeled with
[3H]Ins at 37 °C (5 µCi/A600), and lipid extracts were treated
with PI-specific phospholipase C to get rid of labeled PI, and the GPI
intermediates M0 and M4 were purified from sec53 and
gpi7, respectively, by two rounds of preparative TLC.
Anchor peptides were prepared from [3H]Ins-labeled W303
in the experiment described in Table I. M4 and anchor peptides were
treated with HNO2 to liberate the
acyl-[3H]Ins-P-lipid and
[3H]Ins-P-lipid moieties, respectively (Fig. 1). Samples
were incubated with (+) or without (
) methanolic NH3 to
remove the acyl from the Ins, desalted and separated by TLC (solvent
1), and processed for fluorography. pG1, protein-derived
glycerophospholipid 1; pC1 and pC2, protein-derived ceramides 1 and 2, see Sipos et al. (41). Other results of this same experiment
were described before (Ref. 41, therein Fig. 2).
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Fig. 5.
Analysis of the head group of M4 using HF,
JBAM, and ASAM. gpi7 was labeled with
[3H]Ins at 37 °C; M4 was purified and used to prepare
head groups. Head groups were subjected to HF and
N-acetylation (A); JBAM, then HF, then
N-acetylation (B); HF, then
N-acetylation, then desalting, then JBAM (C); HF,
then N-acetylation, then desalting, then ASAM
(D). The thus generated fragments were separated by paper
chromatography, and radioactivity contained in 1-cm wide strips was
determined through scintillation counting. The position of standards
run in parallel on the same paper are indicated: 2-4,
Manx-GlcNAc-Ins with x = 2, 3, or 4. 0, GlcNAc-Ins.
1,6-glycosidic bonds (Fig. 1). Here this procedure is expected to
produce the labeled fragment
(X-P
?)Man
1,4-GlcN
1,6-[3H]Ins
with X-P- being the substituent in question. The fragment was then either treated with JBAM or control incubated and finally dephosphorylated by HF, N-acetylated, and analyzed by paper
chromatography. As can be seen in Fig. 6,
A and B, the
(X-P
?)Man
1,4GlcN
1,6Ins fragment of M4 is
JBAM-resistant, since successive treatment by acetolysis, JBAM, and
then HF generates Man
1,4GlcN
1,6Ins. The same had previously been
found for CP2 (22). Thus, the difference between the head groups of M4
and CP2 cannot be explained by the presence or absence of an
HF-sensitive substituent on Man1: both lipids have the same classical
Man4-GlcN-Ins carbohydrate core structure, they both
contain HF sensitive groups on Man1 and Man3 (Fig. 1), but they migrate
differently on TLC, and their non-dephosphorylated head groups elute
differently on Dionex HPLC (data presented above and in Refs. 22 and
23). We thus hypothesized that CP2 may contain either additional
HF-sensitive groups on GlcN, Man1, Man2, or Man3 or may contain
additional groups linked through the amino group of the EtN-P on M1. Of
these several theoretical possibilities, the only ones that have been
documented in other organisms are the Man1-P group on the GlcN in
Paramecium aurelia (43) and the EtN-P group on Man2 in
several mammalian GPI proteins, e.g. human erythrocyte
acetylcholinesterase (44), CD52-II (45), and bovine liver
5'-nucleotidase (46). We used limiting HF treatment to test
specifically if CP2 contains an HF-sensitive group on Man2. If we
assume that during HF treatment the EtN-Ps are hydrolyzed in a random
order, we may expect to find some reaction intermediates lacking the
HF-sensitive group on Man3 while retaining EtN-P on Man2 or Man1. When
such intermediates subsequently are treated with JBAM and then are
dephosphorylated to completeness with HF, they should yield
Man2-GlcN-Ins and Man1-GlcN-Ins fragments,
respectively. For preliminary tests, CP2 head groups were first treated
with HF for 0.5, 1, 3, 9, 12, 18, or 28 h, then with JBAM, and
finally with HF for 60 h. These experiments showed that both
Man2-GlcN-Ins and Man1-GlcN-Ins fragments
became visible after 1 h of limiting HF treatment, peaked at
12 h, and remained detectable at all time points up to 28 h.
In quantitative terms it appeared that Man3-GlcN-Ins > Man2-GlcN-Ins
Man1-GlcN-Ins at all
time points. Importantly, treatment of the head group of CP2 with HF
for 12 h yielded substantial amounts of Man2-GlcN-Ins
and Man1-GlcN-Ins (Fig. 6C), whereas the
identical treatment performed with the head group of M4 only yielded
Man1-GlcN-Ins but no Man2-GlcN-Ins (Fig.
6D). This result is compatible with the idea that
gpi7 cells are unable to add an HF-sensitive group onto
Man2 of the GPI core (Fig. 1). It also confirms the presence of an
HF-sensitive group on Man1 of both M4 and CP2.
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Fig. 6.
M4 of gpi7 contains an
HF-sensitive substituent on Man1 but lacks an HF-sensitive substituent
on Man2. CP2 and M4 head groups were obtained from
[3H]Man labeled pmi40 and
[3H]Ins labeled
gpi7, respectively.
A and B, head groups of M4 were subjected to acetolysis
and then either treated with JBAM (B) or left untreated
(A). Finally all products were dephosphorylated with HF,
N-acetylated, and analyzed by paper chromatography.
C and D, head groups were treated for 12 h
with HF, desalted, treated with JBAM, treated with HF for 60 h,
N-acetylated, and finally analyzed by paper chromatography.
Standards 0-4 are Manx-GlcNAc-Ins
(x = 0, 1, 2, 3, 4). Free [3H]Man ran out
of the paper shown in C.
gpi7 Are
Normal--
We looked for additional differences between M4 and CP2 by
analyzing the lipid moiety of M4. M4 is sensitive to GPI-specific phospholipase D (GPI-PLD) and mild base treatment (Fig. 4A, lanes 3-6), suggesting that its lipid moiety consists of Ins-P-DAG. We
previously reported that M4 is resistant to PI-specific phospholipase C
(21). This finding, together with the GPI-PLD sensitivity, can be taken
as an indication for the presence of an acyl moiety attached to the Ins
of M4. We further released the (acyl
)Ins-P-DAG moiety of M4 with
HNO2 as described recently (41). As shown in Fig.
4B, the treatment of purified M4 by HNO2
produced a very hydrophobic species, which migrates very closely to M0,
i.e. the GlcN(acyl
)Ins-P-DAG accumulating in
sec53 (Fig. 4B, lanes 7 and 9) (41). (As reported previously, the presence of GlcN on
these early precursors does not significantly influence their migration in TLC, for discussion see Sipos et al. (41).) Partial
deacylation of the M4-derived lipid moiety by NH3 produced
PI and lyso-PI (Fig. 4B, lane 10). This PI was compared with
pG1, the PI species obtained by HNO2 treatment of
protein-bound GPI anchors from the corresponding wt strain (Fig.
4B, lane 11). The comparison shows that M4 contains a PI
moiety that migrates clearly less than pG1, whereas a lyso-PI of M4
migrates slightly more than the lyso-PI species generated by methanolic
NH3 treatment of anchor peptides (Fig. 4B, lanes
10 and 12). Very similar results had been obtained previously when comparing protein-derived PI moieties with the PI
moieties of M0 from sec53 and of CP2 from gpi8-1
(41). In addition we isolated from
gpi7 the recently
identified GPI intermediates that are obscured in TLC by PI and
inositol phosphoceramides (Ref. 41, therein Fig. 6A), and we
found that they are exactly the same as the corresponding intermediates
from wt cells by all criteria (not shown). Thus, it seems that M4 and
other GPI intermediates of
gpi7 contain the same PI
moiety as early and late GPI intermediates accumulating in other
mutants or in wt cells, and we therefore conclude that the difference
between CP2 and M4 is solely due the difference in their head groups.
gpi7 microsomes, they reproducibly made all
the normal intermediates down to a band comigrating with M4 of
[3H]Ins-labeled
gpi7 cells, but they
consistently failed to make CP2 (Fig. 7).
When the labeled lipid extract was treated with JBAM, most of the band
comigrating with M4 was shifted to a less hydrophilic position, much in
the same way as seen for [3H]Ins-labeled M4 (Fig. 2,
lanes 1 and 2). Thus, the M4 accumulation of
gpi7 can be reproduced in vitro. This result
implies that the Gpi7p present in wt microsomes is functional in
vitro.
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Fig. 7.
Microsomes of gpi7
synthesize M4 and not CP2. Microsomes of W303 wild type or
gpi7 were incubated with 6 µCi of
UDP-[3H]GlcNAc, GDP-Man, tunicamycin, and ATP for 1 h at 37 °C as described (22). The glycolipid products were extracted
and then run on TLC with solvent 2. The extract in lane 3 was first treated with JBAM. On the basis of the preceding analysis the
band denoted with an asterisk can be presumed to be an M4
derivative in which Man4 has been removed.
och1/mnn1 or mnn9 which are totally or
partially deficient in the elongation of N-glycans in the
Golgi, Gpi7p has an estimated mass of 108 and 115 kDa, respectively
(Fig. 8A, lanes 1 and 7). pmi40 has a
ts deficiency in Man biosynthesis that is partial at 24 °C (48). In
pmi40 grown at 24 °C the average mass of Gpi7p is around
150 kDa (Fig. 8A, lane 6). (This suggests that full
elongation of N-glycans is not necessary for Gpi7p function
since, when shifted from 24 to 37 °C, pmi40 cells are
able to make CP2 (23).) Tunicamycin treatment of wt or pmi40
cells resulted in the appearance of a single, relatively sharp band of
an apparent molecular mass of about 83 kDa (Fig. 8A, lanes 3 and 5). The protein could also be deglycosylated to an
apparent molecular mass of 86 kDa by treatment with endoglycosidase H
(not shown). All these data concurrently indicate that Gpi7p contains
several N-glycans that are heavily elongated in the Golgi
but contains no or only few O-glycans. In the cell lysate
Gpi7p was rapidly degraded by an endogenous protease which, however,
could be inhibited by 10 mM EDTA. Gpi7p is associated with
membranes since it could be sedimented by ultracentrifugation of
lysates at 100,000 × g for 60 min (Fig. 8B,
lanes 1 and 2). Gpi7p was neither dissociated from
membranes by NaCl nor sodium carbonate at pH 11, nor urea, but it was
efficiently solubilized by Triton X-100 or SDS (Fig. 8B).
The presence of an N-terminal signal sequence and the large amount of
N-glycans suggested that the hydrophilic N-terminal part of
the protein, which contains 3 of the 5 potential
N-glycosylation sites, would reside on the lumenal or
ectocytoplasmic side of the membrane. We tried to confirm this
orientation by protease protection assays on microsomes. As shown in
Fig. 8C, neither Gpi7p nor Wbp1p, which was used as a
control, were degraded by proteinase K unless microsomes were permeabilized with Triton X-100. Wbp1p has been demonstrated to be a
lumenal ER protein (49), and our result thus shows that the N-terminal
hydrophilic part of Gpi7p is not accessible to protease in these
microsomes. It is noteworthy that proteinase K did not reduce the
molecular mass of Gpi7p, thus indicating that the cytosolic loops
between the predicted transmembrane domains of the C-terminal part of
Gpi7p are not accessible to proteinase K in native microsomes.
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Fig. 8.
Membrane association, orientation, and
localization of Gpi7p. A, exponentially growing cells
were broken with glass beads in TEPI buffer as described (19); lysates
were centrifuged at 10,000 × g for 15 min at 4 °C,
and microsomal pellets were processed for SDS-PAGE. Cells in
lanes 3 and 5 had been grown in 20 µg/ml
tunicamycin (Tm) for 90 min. B-D, exponentially
growing W303 cells were broken with glass beads using the buffers
indicated under "Experimental Procedures," and cell wall debris was
removed by centrifugation at 600 × g. B,
aliquots of cell lysate were incubated for 30 min at 0 °C with 0.5 M NaCl, 0.8 M urea, 1% Triton X-100
(TX-100), 0.1 M Na2CO3,
pH 11, or 1% SDS. Subsequently membranes were sedimented by
ultracentrifugation to get supernatant (S) and pellet
(P) fractions. C, cell lysate was sedimented at
13,000 × g for 15 min, and the membrane pellet was
thoroughly resuspended and digested with 10 or 25 µg/ml proteinase K
(prot K) at 0 °C for 20 min in the presence or absence of
0.5% Triton X-100. D, cell lysates were subjected to
differential centrifugations at 8,000 and 100,000 × g.
These centrifugations generated pellet P8 containing ER, PM, and
vacuolar membranes and pellet P100 which contains Golgi membranes. The
8,000 × g supernatant was also precipitated with
trichloroacetic acid (TCA). In all panels the lanes contain
material derived from 1 A600 of cells except for
lanes 1 and 7 of A which contain 0.3 A600.
gpi7 cells except for a 108-kDa form of Gpi7p (Fig.
9C, lanes 7 and 8). This material seems to be an
ER form in transit to the surface since it was no more detectable if
cells were preincubated with either cycloheximide or tunicamycin (Fig.
9C, lanes 1-6). These data also indicate that mature Gpi7p is relatively rapidly degraded or becomes resistant to extraction with
SDS. Globally these data indicate that the bulk of Gpi7p is exposed at
the cell surface but that a small amount of core-glycosylated material
is found in the ER in transit to the cell surface. For the moment it is
unclear why Gpi7p was completely protected in microsomes, since it has
been claimed that PM does not form closed vesicles upon homogenization
(51). It is conceivable that Gpi7p resides in special PM subdomains
that form closed vesicles upon homogenization or that centrifugation of
microsomes generated protease-resistant membrane aggregates (51).
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Fig. 9.
Gpi7p is localized at the cell surface.
Cells in the early exponential phase growing at 30 °C in YPD were
used. A, intact W303 cells were treated with cysteamine
chloride and then treated with the indicated concentrations of
proteinase K (prot K) exactly as described (65) except that
the EDTA concentration was raised to 20 mM. In lane
6, Triton X-100 was added to 1%. B, W303 cells were
treated with zymolyase 20T at the indicated concentrations.
C, W303 or gpi7 cells were either lysed
directly or after having been incubated for 30 or 60 min at 10 A600/ml in the presence of cycloheximide (200 µg/ml) or tunicamycin (Tm, 20 µg/ml). Cells were lysed
by boiling in sample buffer and processed for SDS-PAGE and Western
blotting with antibodies against Gpi7p, Gas1p, or Wbp1p.
gpi7.
Indeed, by pulse-chase experiments we found that the maturation of GPI
proteins Sag1p and Gas1p was slowed 2-3-fold as compared with wt
cells, whereas the maturation of carboxypeptidase Y proceeded with
normal kinetics (not shown). This indicates that the transport of GPI
proteins in
gpi7 is specifically retarded. Nevertheless,
in rich media
gpi7 cells grow at roughly the same rate as
wt cells. They also incorporate [3H]Ins with the same
efficiency as wt cells.
gpi7. As seen in Table I,
the proportion of ceramides (pC1 and pC2) in anchor peptides from
gpi7 is drastically decreased, whereas the fraction of
DAG-containing lipids ((pG1) is correspondingly increased. It should be
noted that at the time of analysis, i.e. 75 min after
addition of [3H]Ins, the relative amounts of mild
base-sensitive and mild base-resistant anchors are no longer changing
and represent the steady state proportion of these two anchor types
(41, 52). It is important to realize that pG1 also represents a
remodeled form of the anchor lipid in which a long chain fatty acid has
replaced the original fatty acid present in sn-2 of the
glycerol of the CP (Fig. 1). It thus appears that the relative decrease
of ceramide remodeling goes along with a compensatory increase in DAG
remodeling. A relative reduction in ceramide remodeling was also
observed when we compared the efficiency of [3H]Ins and
[3H]dihydrosphingosine ([3H]DHS)
incorporation into GPI proteins. As can be seen in Fig. 10, the ratio of
[3H]DHS/[3H]Ins incorporation into proteins
is much higher in wt than in
gpi7 (Fig. 10, lanes
1-4). The lack of incorporation of [3H]DHS in
gpi7 cannot be explained by an increase of the endogenous production of DHS in
gpi7, since the difference between
wt and
gpi7 persists, even when all endogenous DHS
biosynthesis is blocked by myriocin (Fig. 10, lanes 5 and
6). The defect in remodeling seems to be affecting mostly
the maturation processes in the Golgi and/or PM (Golgi/PM remodeling)
since, as shown in Fig. 10, lanes 7-10, the ratio of
[3H]DHS/[3H]Ins incorporation into proteins
in the
gpi7/sec18 double mutant was the same as in
sec18. Also, when using stringent conditions under which one
observes only ER or only Golgi/PM remodelase (24), remodeling in the ER
appeared relatively normal, whereas remodeling in the Golgi/PM was
reduced (Fig. 10, lanes 11-14, 11', and 12'). The relatively low amount of pC2 in anchor lipids of
gpi7
(Table I) may be a consequence of this relative deficiency of Golgi/PM remodeling, since pC2 type anchors are only generated by the Golgi/PM but not the ER remodelase (24, 41). The relationship between the
specific retardation of GPI protein transport, reduced Golgi/PM remodeling, and increased remodeling toward pG1 is for the moment unclear.
Quantitation of GPI anchor lipids
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Fig. 10.
Ceramide remodeling of GPI anchors is
reduced in gpi7. Cells growing exponentially
in SDCUA were used. Cells were labeled by the addition of either 25 µCi of [3H]DHS (D) or [3H]Ins
(I) to an aliquot of 2.5 A600 of
cells exactly as described (24). Lanes 1-6, precultures and
labelings were at 30 °C. Cells were preincubated with (+) or without
(
) 40 µg/ml of myriocin (myr) for 20 min before addition
of the radiotracers. Lanes 7-10, sec18 or
sec18
gpi7 double mutants were precultured at 24 °C
and preincubated for 10 min at 37 °C before addition of the tracers.
Lanes 11-14, cells were labeled in the presence of
cycloheximide under conditions in which only the Golgi/PM remodelase
(lanes 11 and 12) or only the ER remodelase
(lanes 13 and 14) is probed (Ref. 24, therein
Fig. 7 (Golgi/PM remodelase) and Fig. 8 (ER remodelase)). All samples
were processed for SDS-PAGE under reducing conditions and fluorography.
Lanes 11' and 12' were scanned at an increased
sensitivity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gpi7 cells are hypersensitive to Calcofluor White and
hence have some difficulty in constructing their cell walls. Several reasons can be envisaged. (i) The side chain on Man2 may be important for the interaction of CPs with the transamidase complex and for their
efficient transfer onto proteins. Recent data show that a small
reduction of Gpi8p renders transamidase activity
rate-limiting.3 The synthetic effect of gpi7
mutations with gpi8 mutations suggests that deletion of
GPI7 may have a similar effect. A decreased transamidase activity may particularly affect the anchoring of certain GPI proteins
that have a low affinity for the transamidase even though the global
rate of GPI biosynthesis and [3H]Ins incorporation into
proteins of
gpi7 is not grossly reduced. Thus it is
conceivable that some GPI proteins important for cell wall architecture
are lacking in
gpi7. (ii) The side chain on Man2 may
serve as an attachment point for the covalent linkage of
1,6-glucans
to the anchor moiety of cell wall proteins although a recent analysis
of the linkage region between the GPI anchor remnant and
1,6-glucans
rather showed a direct glycosidic linkage between Man1 and the
1,6-glucan (6). (iii) The side chain may serve as a recognition
signal for enzymes or proteins that facilitate the packaging of GPI
proteins into vesicles, for remodelases that exchange their lipid
moieties, or for hydrolases or transglycosidases that remove parts of
the GPI anchor of cell wall proteins and hook the GPI remnant onto
1,6-glucans (4, 5).
gpi7, whereas remodeling toward pG1 is increased whereby it is not clear if pG1 remodeling is increased
because ceramide remodeling is decreased or if ceramide remodeling is
decreased because pG1 remodeling is increased. Moreover, the
relationship of the alteration of GPI remodeling with the other
phenotypic changes of
gpi7can be explained in several
ways. (i) Previous studies showed that remodeling toward pG1 occurs in
the ER and that retention of GPI proteins in the ER in secretion mutants maintains a high pG1/pC1 ratio on these proteins (41). Thus, if
the substituent on Man2 of GPI anchors is important for efficient
packaging of GPI proteins into transport vesicles, the delay in export
of GPI proteins out of the ER may give the ER remodelase generating pG1
prolonged access to the GPI proteins and may thus cause a relative
increase of pG1. (ii) The side chain on Man2 may serve as a recognition
signal for Golgi/PM remodelase. (iii) We also considered the
possibility that Gpi7p itself may be a Golgi/PM remodelase. This latter
hypothesis would not directly explain why
gpi7 cells
cannot attach the HF-sensitive substituent onto Man2 and would imply
that the addition of this side chain somehow is directed by the prior
attachment of a ceramide moiety. This, however, is clearly not the
case, since CP2 also contains the HF-sensitive side chain on Man2,
although its lipid moiety consists of DAG (23). Thus we believe that
the reduced Golgi/PM remodeling of GPI proteins in
gpi7
is secondary to the lack of a substituent on Man2.
gpi7 incorporates
[3H]Ins at a normal rate into proteins suggesting that
the transamidase is perfectly able to transfer M4. Thus, the side chain
on Man2 may normally not be added to GPI proteins or only be added
after GPI proteins arrive at the cell surface. The M4 hypothesis,
however, does not explain why M0 and M4 accumulate in
gpi7, whereas M0, M4, and CP2 remain undetectable in wt
cells (Fig. 2, lanes 1 and 2) or why
gpi8-1, deficient in the transfer of GPIs onto proteins, accumulates CP2 (19, 21). It also fails to explain the delayed maturation of GPI proteins and the reduced rate of GPI remodeling observed in
gpi7. To save the M4 hypothesis, the
accumulation of GPI intermediates in
gpi7 could be
rationalized by assuming that the substituent on Man2 serves to mark
supernumerary GPIs for degradation, but also this assumption does not
explain the observed accumulation of CP2 in gpi8. Thus,
although our results raised the possibility that M4 is the
physiological GPI lipid for GPI anchoring, this M4 hypothesis leaves
many results unexplained and the data are more easily explained by our
original CP2 hypothesis. For one, the synchronous accumulation of M4
and CP2 in all our gpi8 mutants argues that M4 is not a
better substrate for the transamidase than CP2. CP2 may physiologically
be produced by the small amount of Gpi7p in the ER (Fig.
9C). Alternatively, it is conceivable that M4 is transported
from the ER to the PM, is converted there to CP2, and is then
transported back to the ER by some not yet elucidated mechanism. In
this context it is noteworthy that the biosynthesis of GPIs by wt
microsomes in vitro produces CP2 in good yield,
i.e. the in vitro system adds the substituent on
Man2. This in vitro system does not contain cytosol nor GTP
and hence should not support vesicular transport from ER- to
Golgi-derived microsomes (59). It is possible, however, that GPI lipids
are transported between microsomes or membrane fragments by means of
lipid transfer proteins or through direct contact between membranes. It
also can be envisaged that juxtaposition of membranes allows enzymes
present in one membrane to work on lipids in another membrane. The same
mechanisms may also operate in intact cells. Clearly the identity of
the physiological GPI lipid substrate of the transamidase will have to
be established by further experiments.
GPI7 has homologues in other species
It is interesting that subfamily members belonging to different species are more closely related to each other than family members belonging to a single species. This can be seen when comparing the pairwise alignment scores among the three ORFs of S. cerevisiae or the three ORFs of Schizosaccharomyces pombe with the scores among subfamily members (Table II). In evolutionary terms this suggests that the divergence of these three subfamilies occurred earlier than the separation of the lineages leading to S. cerevisiae, S. pombe, and Caenorhabditis elegans. This implies that the HF-sensitive group on Man2 is of very ancient origin. GPI7 bears no resemblance with PIG-F, a mammalian gene encoding for a highly hydrophobic membrane protein involved in the addition of EtN-P to Man3 (10). The exact role of PIG-F has not yet been elucidated.
It will be interesting to find the human homologues of GPI7.
It may be that this gene, as in S. cerevisiae, plays a more
dispensable role in GPI anchoring than the enzymes involved in the
elaboration of the carbohydrate core structure such as for instance
PIG-A/GPI3/CWH6/SPT14 (7). Thus, although deficiencies in
PIG-A are only acquired by somatic cells, deficiencies in
the human GPI7 homologue may be transmittable through the
germ line as well.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. N. Rao Movva for myriocin; and Dr. R. Fuller, Dr. Y. Jigami, Dr. S. Emr, Dr. M. Aebi, Dr. M. Payton, Dr. H. Riezman, and Dr. R. Schekman for antibodies and yeast strains. We are grateful to Dr J. van Oostrum (Novartis, Basel, Switzerland) for giving us access to and helping with the Dionex HPLC. We especially thank Dr. S. Munro, MRC, Cambridge, UK, for pointing out the homology between GPI7 and the phosphodiesterase family.
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FOOTNOTES |
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* This work was supported by Grant 3100-032515 from the Swiss National Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Université de Caen, IRBA, Laboratoire de
Microbiologie de l'Environnement, Caen, France.
§ Present address: c/o R. S. Fuller, University of Michigan Medical Center, Dept. of Biological Chemistry, Ann Arbor, MI 48109-0606.
¶ Present address: c/o H. Pelham, MRC, Laboratory of Molecular Biology, Cambridge, UK.
To whom correspondence should be addressed: Institute of
Biochemistry, Chemin du Musée 5, CH-1700 Fribourg, Switzerland. Tel.: 41 26 300 8630; Fax: 41 26 300 9735: E-mail:
andreas.conzelmann{at}unifr.ch.
** Present address: Plant Cell Biology, Research School of Biological Sciences, Australian National University, Canberra ACT 2601, Australia.
2 I. Flury, unpublished observations.
3 I. Imhof, U. Meyer, A. Benachour, I. Flury, E. Canivenc-Gansel, C. Vionnet, and A. Conzelmann, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
GPI, glycosylphosphatidylinositol;
ASAM, A. satoi
-mannosidase;
CP, complete precursor;
DAG, diacylglycerol;
DHS, dihydrosphingosine;
EtN-P, ethanolamine phosphate;
GPI-PLD, GPI-specific phospholipase D;
Ins, myo-inositol;
JBAM, jack
bean
-mannosidase;
Man, mannose;
ORF, open reading frame;
pC1 and
pC2, protein-derived Ceramides 1
and 2;
pG1 protein-derived
Glycerophospholipid 1, PI,
phosphatidylinositol;
PM, plasma membrane;
ts, thermosensitive;
wt, wild type;
PAGE, polyacrylamide gel electrophoresis;
PIPES, 1,4-piperazinediethanesulfonic acid;
nt, nucleotide;
PCR, polymerase
chain reaction;
kb, kilobase pair(s);
HPLC, high pressure liquid
chromatography.
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REFERENCES |
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