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J. Biol. Chem., Vol. 275, Issue 32, 24458-24465, August 11, 2000
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From the Institute of Biochemistry, University of Fribourg,
CH-1700 Fribourg, Switzerland
Received for publication, May 4, 2000
MCD4 and GPI7 are
important for the addition of glycosylphosphatidylinositol (GPI)
anchors to proteins in the yeast Saccharomyces cerevisiae.
Mutations in these genes lead to a reduction of GPI anchoring and cell
wall fragility. Gpi7 mutants accumulate a GPI lipid
intermediate of the structure
Man The structural analysis of
GPI1 anchors revealed that
different organisms add different side chains to the universally
conserved Man Here we investigate whether YLL031c plays any role in GPI biosynthesis
and, specifically, in the addition of EtN-P- groups to GPI structures.
Strains, Growth Conditions, and Materials--
S.
cerevisiae strains were FBY413 (MATa ura3-52
leu2-
Materials were obtained from the sources described recently (4).
Oligonucleotides were from Microsynth GmBH (Balgach, Switzerland), Quantazyme ylg from Quantum Biotechnologies Inc. (Montreal, Canada), Calcofluor White (CFW) from Sigma, and antibodies to carboxypeptidase Y
from Molecular Probes, Inc. (Eugene, OR). Rabbit antibodies to Yap3p
and Cwp1p were kindly donated by Dr. Y. Bourbonnais (Université Laval, Québec, Canada) and Dr. H. Shimoi (National Institute of
Brewing, Higashihiroshima, Japan), respectively. Rabbit antibodies to
Och1p and Wbp1p were gifts from Dr. Y. Jigami (National Institute of
Bioscience and Human Technology, Ibarasaki, Japan) and Dr. M. Aebi
(Mikrobiologisches Institut, ETH Zürich, Switzerland), respectively. A mouse monoclonal anti-Myc antibody, clone 9E10, was obtained from Babco (Richmond, CA), and goat anti-mouse
IgG-peroxidase conjugate was from Sigma.
Conditional Expression of YLL031c and MCD4--
Conditional
expression of YLL031c was achieved by the insertion of the
GAL1,10 promoter in front of the chromosomal YLL031c gene as
described (13). Briefly, the HIS3 marker flanked by the
GAL1,10 promoter was PCR-amplified using pTL26 as template and the following two adapter primers: 031GalFor
(5'-AAGATCAAAAAAGGAATAGAAGCATATGTTTTAAGGGCAACGCCGctcttggcctcctctag-3') with 17 nt of homology to the pTL26 vector (lowercase) and 45 nt of
homology to a 5'-flanking sequence of YLL031c (uppercase) and
oligonucleotide 031GalRev
(5'-AAGAATCGACTTTTTAATTGTCTTTTCATCCATATTACGGGAGCTcgaattccttgaattttcaaa-3') with 45 nt of homology to the 5'-end of YLL031c ending 12 nt
upstream of the start codon (boldface type) and 21 nt of homology to
the pTL26 vector (lowercase type). This PCR-generated DNA fragment was
used to transform the strains FCEN010a and FBY413, yielding FBY1103 and
FBY1102, respectively. Correct targeting of the inserted promoter was
verified by whole yeast cell PCR (14), using primers A3Gal
(5'-gagcagttaagcgtattactg-3') and 031A4 (5'-catcaatgaaagtcggtaagg-3') yielding a 0.7-kilobase pair DNA fragment.
The genomic MCD4 was placed under the control of the
GAL1,10 promoter by the same strategy as described above for
YLL031c, using the following two adapter primers for PCR amplification from the pTL26 as the template: 165GalFor
(5'-GAACCGTTCTTTACTATATATTCAACAACCCATCTTCGACCAAAGctcttggcctcctctag-3') with 17 nt of homology to the pTL26 vector (lowercase type) and 45 nt
of homology to the MCD4 flanking sequence (uppercase type) and oligonucleotide 165GalRev
(5'-GACACCAACAGCCAGAAGCGTCGTTCTGGTTTTGTTCCACATTTTcgaattccttgaattttcaaa-3') with 45 nt of homology to MCD4 ending 3 nt upstream of
the start codon (boldface type) and 21 nt of homology to the pTL26
vector (lowercase type). This PCR-generated DNA fragment was used to transform the strain FCEN010a. Correct targeting of the
HIS3-marked GAL1,10 promoter was verified by
whole yeast cell PCR using primers A3Gal (5'-gagcagttaagcgtattactg-3')
and 165A4 (5'-agcaatcatagcaacatgacc-3') yielding a 0.5-kilobase pair
DNA fragment.
Disruption of YLL031c--
For deletion of YLL031c, a short
flanking homology (SFH) replacement cassette was synthesized by PCR
amplification of the KanMX4 module of pFA6a-KanMX4 (15) with the
following primers: primer 031-S1
(5'-GATGAAAAAATAATATACAAATCGCGAATAAAGAAATTTCAAcgtacgctgcaggtcgac-3') with 42-nt homology to amino acids 16-29 of YLL031c and 18-nt homology
to the KanMX4 module (lowercase type) and primer 031-S2 (5'-
TATAGTATATTTGTAAGTAAAGAGTGGAAATGAAGTTCGTCATTatcgatgaattcgagctcg-3') with 19 nt of homology to the KanMX4 module (lowercase type) and 44 nt
of homology to a sequence near the 3' end of YLL031c (uppercase type)
comprising the stop codon (boldface type). Since gene replacement with
this SFH cassette proved to be difficult, the SFH cassette was used as
a template to construct a long flanking homology cassette. This long
flanking homology replacement cassette was synthesized as described
(16). In a first step, the primer pair K1
(5'-CATGGTACAATTGCAAAGT-3') and L2
(5'-TTGAAATTTCTTTATTCGCGATTTGTATATTAT-3') as well as the primer pair L3
(5'- AATGACGAACTTCATTTCCACTCTTTACTTACAAATATAC-3') and A4
(5'-GCTGCAGAAAAGAGATGC-3') were used to amplify two fragments of
genomic DNA corresponding to the promoter region including the first 29 codons of the open reading frame and the terminator region,
respectively. The 5'-parts of primers L2 and L3 were designed to
anneal, respectively, to the 5'- and 3'-ends of the SFH replacement cassette, which are homologous to YLL031c. In the second step, a PCR
was performed using the SFH cassette as template and the two
PCR-generated fragments from the first step as primers, yielding a
replacement cassette with long flanking regions having homology to the
YLL031c gene. The long flanking homology cassette was used to transform
the diploid WT strain FY1679, homozygous for YLL031c. The correct
targeting of the KanMX4 replacement cassette to the YLL031c locus was
verified by whole yeast cell PCR using primer K1, primer 031-A5
(5'-catcaatgaaagtcggtaagg-3') annealing inside the YLL031c coding
sequence, and the K2 primer of the KanMX4 module (5'-gtcgcacctgattgcccg-3'), yielding a 900-bp DNA fragment for the
disrupted gene and a 748-bp DNA fragment for the WT YLL031c gene.
Tagging of YLL031c--
A Myc tag was inserted at the C terminus
of the genomic copy of YLL031c by homologous recombination with an
insertion cassette containing the Myc tag and the selectable KanMX6
marker as described (17). The insertion cassette was obtained by using
plasmid pFA6a-13Myc-kanMX6 (17) as template and the oligonucleotides F2
and R1 as target gene-specific primers (F2,
5'-AGTGGGAGATTGATTAAGCACATAAATGACATTTTTTGGAAAcggatccccgggttaattaa-3', with 20-nt homology to the pFA6a-13Myc-kanMX6 sequence (lowercase type) and 42-nt homology to the sequence of YLL031c immediately upstream of the stop codon (uppercase type), including the codon of the
last amino acid (boldface type); R1,
5'-ATATATAGTATATTTGTAAGTAAAGAGTGGAAATGAAGTTCGgaattcgagctcgtttaaac-3' with 42-nt homology to the genomic sequence of YLL031c
immediately downstream of the stop codon (uppercase) and 20-nt homology
to the pFA6a-13Myc-kanMX6 plasmid sequence). This PCR fragment was used
to transform the WT strain FBY413, yielding FBY1107. The correct
targeting of the PCR-made module was verified by whole yeast cell PCR
using the primer K2 of the kanMX module and oligonucleotide C1-Myc
(5'-CTGACACTGTGGTCACAGCC-3'), producing a 1577-nt fragment.
Cellular Localization and Protease Sensitivity of YLL031c-Myc
Protein--
The subcellular localization of YLL031c-Myc was
determined essentially as described (18). Briefly, 100 optical density
units of exponentially growing FBY1107 cells were washed with 10 mM NaN3 and converted to spheroplasts by
incubation for 1 h at 30 °C with Quantazyme in 1.4 M sorbitol, 10 mM NaN3, 50 mM K2HPO4, pH 7.5, 40 mM 2-mercaptoethanol. Spheroplasts were resuspended in
lysis buffer (20 mM HEPES-KOH, pH 6.8, 150 mM
potassium acetate, 250 mM sorbitol, 1 mM
magnesium acetate, 20 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml
antipain, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin) and homogenized
by 20 strokes in a Dounce homogenizer. The crude lysate was centrifuged
twice at 1000 × g for 5 min to remove unlysed spheroplasts. The cleared supernatant was then centrifuged at 13,000 × g for 15 min to generate pellet P13 and
supernatant S13. S13 was centrifuged at 100,000 × g
for 60 min in a Sorvall AH-650 swing out rotor to generate pellet P100
and supernatant S100. Pellet fractions were dissolved and denatured in
high urea buffer (8 M urea, 5% SDS, 200 mM
Tris-HCl, pH 6.8, 20 mM EDTA, bromphenol blue, 15 mg/ml
dithiothreitol) by sonication and incubation at 95 °C for 5 min. The
S100 fraction was trichloroacetic acid-precipitated before denaturation
in high urea buffer.
For probing the surface exposure of YLL031c-Myc, exponentially growing
FBY1107 cells were converted to spheroplasts as described above but
using zymolyase-20T. Spheroplasts were either mock-treated or treated
with proteinase K in the presence or absence of 0.5% Triton X-100 for
30 min on ice. Phenylmethylsulfonyl fluoride was then added to a final
concentration of 4 mM, and samples were kept on ice for 15 min. Finally, spheroplasts 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. YLL031c protease sensitivity in microsomes was examined by
protease K digestion essentially as described (8), except that the
membrane pellet was resuspended in 600 µl of lysis buffer before
protease digestions. All samples were denatured during 5 min at
95 °C in reducing sample buffer containing 20 mM EDTA
and analyzed by SDS-polyacrylamide gel electrophoresis in 6-10% gels
(19) followed by Western blotting.
Miscellaneous Methods--
Western blots were revealed using the
chemiluminescence ECL kit from Amersham Pharmacia Biotech.
Previously described procedures were used to label cells with
[2-3H]Ins and for lipid extraction (20).
In vitro biosynthesis of GPIs with microsomes was performed
as described previously (4), with the exception that spheroplasts were
sometimes prepared using Quantazyme ylg instead of zymolyase-20T. Desalted lipid extracts were analyzed by ascending TLC using solvent 1 (CHCl3/CH3OH/H2O, 10:10:3, v/v/v)
or solvent 2 (chloroform/methanol/0.25% KCl, v/v/v). Radioactivity was
detected and quantitated by two-dimensional radioscanning (LB 2842;
Berthold AG, Regensdorf, Switzerland). Thereafter, TLC plates were
sprayed with EN3HANCE and exposed to film (X-Omat; Eastman
Kodak Co.) at
Soluble head groups were obtained from purified radiolabeled
glycolipids through limiting methanolic NH3 deacylation
(21) followed by PI-PLC treatment, for which the lipid extracts were dissolved in 20 mM Tris-HCl, pH 7.5, 0.2 mM
EDTA, 20% 1-propanol. Incubations with PI-PLC were for 16 h at
37 °C. Nonhydrolyzed GPIs were removed by butanol extraction. The
water-soluble head groups were treated with JBAM (0.5 units) or
A. satoi Characterization of YLL031c--
YLL031c was disrupted in a
diploid strain by the replacement of most of the open reading frame by
the KanMX4 kanamycin resistance gene. This deletion strain was
constructed because in the YLL031c deletion strain produced in the
EUROFAN project, part of YLL030c has been deleted along with YLL031c
(24). Sporulation and dissection of tetrads yielded in all cases two
growing colonies per tetrad. None of these colonies were
kanamycin-resistant, indicating that YLL031 is essential. A partial
depletion of YLL031 was achieved by the insertion of the
glucose-repressible GAL1,10 promoter immediately upstream of
the genomic YLL031c. When such cells, growing in liquid culture, were
shifted from galactose to glucose, thus allowing for the partial
depletion of the YLL031c protein, their doubling time increased, after
24 h, to 3.5 h, and cells thereafter continued to grow
at this rate, whereas WT cells grew on glucose with a doubling time of
1.3 h. When the same experiment was done in a Depletion of YLL031c Leads to the Accumulation of Immature GPI
Proteins--
If Gas1p does not receive a GPI anchor in the ER, it
fails to be transported to the Golgi (25), because, as shown by
in vitro experiments using a vesicle budding assay,
nonanchored Gas1p is not packaged into COPII-coated transport
vesicles budding off the ER (26). Therefore, the characteristic mass
increase due to elongation of N- and O-glycans in
the Golgi does not take place, and the maturation of the immature
105-kDa ER form into the mature 125-kDa form is delayed. GPI anchoring
deficiencies also can lead to the relative depletion of the mature
forms of Gas1p and other GPI proteins (27). Using Western blotting and
antibodies to detect well characterized GPI proteins, we could observe
a significant depletion of mature GPI proteins upon depletion of
YLL031c. As can be seen in Fig. 3, while
in WT cells the carbon source affects the amount of Gas1p only
moderately, cells partially depleted of YLL031c by growth on glucose
contain much less of the mature 125-kDa form of Gas1p than cells grown
on galactose. This phenomenon is even more drastic when YLL031c
depletion occurs in a Depletion of YLL031c Leads to the Accumulation of Abnormal GPI
Lipids--
When WT cells are metabolically labeled with
myo-[3H]inositol
([3H]Ins), their lipid extracts contain various forms of
labeled phosphatidylinositol and inositolphosphorylceramide, but the
GPI intermediates remain undetectable. GPI intermediates have, however, been found to accumulate in several gpi mutants. As can be
seen in Fig. 4, A and
B, YLL031c-depleted cells accumulate the abnormal lipids
031a and 031b, which are present neither in WT nor in previously described gpi mutants. Lipid 031a migrates quite differently
from M2*, the most polar lipid accumulating in gpi10-1,
which has the structure
Man
031a and 031b are less polar than M4, suggesting that they have smaller
head groups and are earlier intermediates of GPI biosynthesis than M4.
It therefore is not unexpected that the accumulation of 031b is
epistatic to the accumulation of M4 both in vitro and in vivo. Indeed, the combination of
Structural Characterization of Lipids 031a and
031b--
YLL031c-depleted cells were labeled with
[3H]Ins, and lipids 031a and 031b were purified by
preparative TLC. Their structural analysis yielded the following
information. Both lipids are effectively cleaved by GPI-specific
phospholipase D, indicating that they are GPIs. They are totally
resistant to bacterial, phosphatidylinositol-specific phospholipase C,
suggesting that they contain a protecting acyl group on the Ins. Their
label becomes entirely hydrophilic upon deacylation by mild alkaline hydrolysis.
Analysis of the hydrophilic head group of lipid 031b indicates that it
contains a Man4-GlcN-Ins core structure (Fig.
5A), that two or three of its
mannoses can be removed by JBAM (Fig. 5C), whereas the same
enzyme removes all four mannoses from the HF dephosphorylated head
group (Fig. 5D). If the HF-treated core structure is treated
with Aspergillus satoi
Analysis of the hydrophilic head group of lipid 031a indicates that it
contains a Man4-GlcN-Ins core structure (Fig.
6A) and that all of its four
mannoses can be removed by JBAM not only after but also before HF
dephosphorylation (Fig. 6, C and B). These data
strongly suggest that lipid 031a has the structure Man Subcellular Localization of YLL031c--
YLL031c predicts a
translation product of 116 kDa. To localize the YLL031c protein, the
endogenous, chromosomally encoded gene was modified by the insertion of
a Myc tag at its C terminus. This YLL031c-Myc translation product
(including the N-terminal signal sequence) has a predicted molecular
mass of 136 kDa. The corresponding protein was detected in Western
blots as a major band at about 136 kDa plus a heterogeneously
glycosylated smear with another distinct band at about 300 kDa. The
cells containing the tagged YLL031c grew at the same rate as WT cells
and were completely resistant to CFW, indicating that the tagged
version of the protein is functional. As shown in Fig.
7B, when intact spheroplasts
were treated with variable amounts of zymolyase and proteinase
K, YLL031c-Myc as well as ER proteins such as Wbp1p and Gpi8p remained
intact, whereas Gas1p was strongly diminished, as expected for a
surface protein and as described before (8). Gpi7p could not be
detected on any blot in this experiment (not shown) because of its
previously reported susceptibility to zymolyase (8). When
spheroplasts were prepared with Quantazyme, both YLL031c and Gpi7p were
preserved, but Gpi7p was readily destroyed by proteinase K, whereas
YLL031c-Myc was completely resistant (not shown). Thus, unlike Gpi7p,
YLL031c-Myc does not reside at the cell surface. Differential
centrifugation of microsomes indicated that YLL031c-Myc cofractions
with ER markers Wbp1p and Gpi8p, since it could be found mainly in P13,
which is enriched in ER and plasma membrane (Fig. 7A). Minor
amounts were present in P100, a fraction that is enriched in the Golgi
marker Och1p and is largely free of Gpi8p and Wbp1p (Fig. 7). The
existence of the 300-kDa form is indicative of massive glycan
elongation in the Golgi on part of YLL031c-Myc. Since only little
YLL031c-Myc is found in this organelle at steady state, it may be that
the protein is recirculating between Golgi and ER as has been reported
for other proteins such as Emp47p, Sec12p, and Sed5p. When microsomes
were incubated with proteinase K, even low concentrations of protease (10 µg/ml) readily destroyed the immunoreactivity of YLL031c-Myc (Fig. 7C). In contrast, Gpi8p, which has a large N-terminal
luminal and a small C-terminal cytosolic domain of about 14 amino acids was slightly reduced in size but not destroyed by proteinase K. In view
of the massive glycosylation of part of YLL031c-Myc and the presence of
an N-terminal hydrophobic domain that qualifies as potential signal
sequence, we interpret these findings in the sense that the N-terminal
part of YLL031c containing six N-glycosylation sites is
oriented luminally. The fact that proteinase K treatment of microsomes
does not generate any immunoreactive low molecular weight product
suggests that the C-terminal Myc tag is exposed cytosolically or that
YLL031c-Myc contains a proteinase K-sensitive site close to its C
terminus. The membrane topology of YLL031c thus appears to be the same
as in the homologous Gpi7p (8).
In this study, we investigate the potential function of YLL031c in
GPI anchoring. As shown in Fig. 8,
YLL031c predicts a membrane glycoprotein of 1017 amino acids with an
N-terminal hydrophilic domain and a C-terminal hydrophobic sequence
containing numerous potential transmembrane domains, a feature that
initially led to its classification as potential facilitator of
membrane permeation (32).
YLL031c Belongs to a Novel Family of Membrane Proteins Involved
in the Transfer of Ethanolaminephosphate onto the Core Structure of
Glycosylphosphatidylinositol Anchors in Yeast*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-2[NH2-(CH2)2-PO4
]Man1-2Man1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]inositol-PO4-lipid, which, in comparison with the complete GPI precursor lipid CP2, lacks an HF-sensitive side chain on the 1-6-linked mannose.
In contrast, mcd4-174 accumulates only minor amounts of
abnormal GPI intermediates. Here we investigate whether YLL031c, an
open reading frame predicting a further homologue of GPI7
and MCD4, plays any role in GPI anchoring. YLL031c is an
essential gene. Its depletion results in a reduction of GPI anchor
addition to GPI proteins as well as to cell wall fragility.
YLL031c-depleted cells accumulate GPI intermediates with the
structures
Man1-2Man1-2Man1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]inositol-PO4-lipid and
Man1-2Man1-2Man1-6Man1-4GlcN1-6[acyl]inositol-PO4-lipid. Subcellular localization studies of a tagged version of YLL031c suggest that this protein is mainly in the ER, in contrast to Gpi7p,
which is found at the cell surface. The data are compatible with the
idea that YLL031c transfers the ethanolaminephosphate to
the inner 1-2-linked mannose, i.e. the group that links
the GPI lipid anchor to proteins, whereas Mcd4p and Gpi7p transfer ethanolaminephosphate onto the 1-4- and 1-6-linked
mannoses of the GPI anchor, respectively.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-2[NH2-(CH2)2-PO4]Man1-2Man1-6Man1-4GlcN1-6inositol-PO4-lipid core structure (Fig. 1) (1). In particular, human and all other mammalian cells invariably add an EtN-P group onto Man1 (Fig. 1), and in a few cases they also add an
EtN-P group to Man2 (for a review, see Ref. 2). Although no EtN-P side
chains had been found in a study of a pool of protein-derived GPI
anchors of Saccharomyces cerevisiae (3), it recently was
shown that the GPI intermediate M2* accumulating in a gpi10
mutant has the structure
Man1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]inositol-PO4-lipid (4). Moreover, the complete GPI anchor precursor lipid CP2 that can be
observed in mutants such as pmi40 or in transamidase mutants
(5-7) contains phosphodiester-linked side chains on both Man1 and Man2
(4, 8). This suggests that the side chains on Man1 and Man2 may have
been invented before the separation of the lineages leading to present
day yeast and mammals. Recent studies have implicated MCD4
and GPI7 in the transfer of EtN-P groups onto GPI structures
in yeast. MCD4 is essential, and mcd4-174 cells
show an almost complete defect in GPI anchor addition to proteins at
the restrictive temperature (9, 10). Deletion of PIG-N, a mammalian
homologue of MCD4, leads to a reduced surface expression of
GPI proteins and loss of an enzyme activity that adds EtN-P to Man1
(11). GPI7 is not essential, but its deletion slows the GPI
anchor addition to newly synthesized proteins, produces cell wall
fragility, and blocks biosynthesis of GPI lipids at a premature stage.
Of the several abnormal GPI lipids accumulating in
gpi7 cells, even the most complete one lacks
the phosphodiester-linked substituent on Man2, a finding that relates
GPI7 to the addition of a side chain to Man2 (8). The idea
that MCD4, GPI7, and their homologue YLL031c may
all be involved in the transfer of EtN-P groups has been fostered by
their pronounced homology with bacterial, viral, plant, and mammalian
phosphodiesterases, phosphatases, and pyrophosphatases (8, 10).
View larger version (14K):
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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 are used here to designate the
1-4-linked, 1-6-linked, and 1-2-linked mannoses,
respectively. 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
1 trp1-63
his3-200), FBY1102 (MATa ura3-52
leu2-1 trp1-63 his3-200 yll031c::HIS3-pGAL1, 10-YLL031c), FCEN010a (MATa
ura3-52 trp1-63 his3-200
YNR019w::KanMX4), FBY1103 (MATa ura3-52
trp1-63 his3-200 yll031c::HIS3-pGAL1,10-YLL031c
YNR019w::KanMX4), FBY1104 (MATa ura3-52
trp1-63 his3-200
mcd4::HIS3-pGAL1,10-MCD4 YNR019w::KanMX4), FBY182 (MAT ade2-1 ura3-1 leu2-3,112 his3-11,15
gpi7::KanMX4), FBY1106 (MATa
yll031c::HIS3-pGAL1, 10-YLL031c gpi7::KanMX4 leu2 ura3 his3), FY1679 (MATa/ ura3-52/ura3-52
leu2-1/+ trp1-63/+ his3-200/+), FBY1105 (MATa/
ura3-52/ura3-52 leu2-1/+ trp1-63/+ his3-200/+ YLL031c::KanMX4/YLL031c),
FBY1107 (MATa ura3-52 leu2-1 trp1-63
his3-200 yll031c::YLL031c-13Myc-KanMX6),
FBY122 (MATa ade2-1 ura3-1 leu2-3,112 his3-11,15 trp1-1
gpi8-1 gpi7-1), FBY169 (MAT ade2-1
can1-100 ura3-1 leu2-3,112 his3-11,15 trp1-1 gpi10-1), and
HMSF176 (MATa sec18-1). Maintenance and growth conditions have been described (7). CFW-containing plates were made as described
(12), with the exception that a 100 mg/ml filter-sterilized CFW stock
solution was used.
80 °C.
-mannosidase (5 microunits) as described (22).
HF dephosphorylation was done as described (23). The generated
fragments were analyzed by paper chromatography in
methylethylketone/pyridine/H2O (20:12:11) as described
(23). Before paper chromatography the products were
N-acetylated and desalted over mixed bed ion exchange resin AG-501-X8 (Bio-Rad). Radiolabeled chromatography standards were obtained as described (8).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gpi7 background, the doubling time of
YLL031c-depleted cells, after 24 h on glucose, had risen to
7.7 h, while gpi7 mutants grew as fast as
WT cells. Thus, the deletion of GPI7 synthetically enhances
the growth retardation caused by depletion of YLL031c. The reduced
growth rate of YLL031c-depleted cells could also be observed on YPD
plates, and this effect was significantly enhanced by the presence of
CFW at a concentration that did not affect growth of WT cells but
totally blocked the growth of gpi7 cells (Fig.
2). This result indicates that partial
depletion of YLL031c leads to cell wall fragility, a phenotype that is
commonly observed in mutants affected in the GPI biosynthesis
pathway.
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Fig. 2.
Characterization of YLL031c. From cell
suspensions containing 2.5 × 107 cells/ml, we
prepared sequential 1:10 dilutions. 3-µl aliquots of the various
dilutions were spotted on agar plates containing either YPD medium
(A) or YPD with 1.5 mg/ml of CFW (B). Plates were
photographed after 3 days at 30 °C. The amounts of YLL031c protein
and Gpi7p in the various strains are indicated on the right.
+, normal or elevated due to induction of GAL1,10 promoter;
, depleted due to repression of GAL1,10 promoter; ø = non existent due to deletion of the gene.
gpi7 background. Other
GPI proteins such as Yap3p and Cwp1p are also affected. Although the
details of the biosynthesis of these proteins have not been reported
and part of their mature forms gets covalently attached to the cell
wall, the mass of their ER forms can be inferred form the peptides
accumulating in sec18, a mutant that blocks the protein
traffic from ER to Golgi at 37 °C (28, 29). YAP3 predicts
a 60-kDa translation product, but upon shift to 37 °C, sec18 cells rapidly accumulate lower molecular weight forms
(Fig. 3). Similar forms also accumulate upon depletion of YLL031c,
suggesting a maturation defect of these proteins due to a delay in GPI
anchor addition. Cwp1p is a cell wall protein that can be released as soluble 55-60-kDa protein by 
-glucanase (30, 31). There are also
intracellular, detergent-soluble forms of Cwp1p of 48 and 58 kDa, and
these forms are drastically increased when the secretory pathway is
blocked for 2 h at 37 °C in sec18 (Fig. 3).
In YLL031c-depleted cells, these detergent-soluble forms are severely
diminished. This also can be taken as evidence for some disturbance of
GPI protein maturation, although in this case the protein does not seem
to accumulate but may be rapidly degraded. In contrast,
YLL031c-depleted cells do not accumulate the typical ER proform p1 of
carboxypeptidase Y, a vacuolar hydrolase, nor do they show a thinning
of mature carboxypeptidase Y (Fig. 3). This suggests that the
maturation defect of YLL031c-depleted cells affects only GPI
proteins.
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Fig. 3.
Depletion of YLL031c leads to a depletion of
mature and accumulation or depletion of immature forms of GPI
proteins. Cells indicated at the top were grown for at
least four generations at 30 °C on either Gal or Glc except for
sec18 cells, which were grown at 24 °C but were shifted
to 37 °C 2 h before extraction. Exponentially growing cells
were lysed by boiling for 5 min in reducing sample buffer and processed
for SDS-polyacrylamide gel electrophoresis on 7.5 or 10% gels (19)
followed by Western blotting with antibodies against Gas1p, Yap3p,
Cwp1p, or carboxypeptidase Y (CPY). The expected amounts of
Gpi7p and YLL031c protein in these cells are indicated as in Fig.
2.
1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]Ins-PO4-lipid (Fig. 4B, lanes 3 and 4)
(4). The more polar lipid 031b has a lower RF than
M2* but a higher RF than M4, the GPI lipid that
accumulates in gpi7 cells and has the
structure Man1-2[NH2-(CH2)2-PO4]Man1-2Man1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]Ins-PO4-lipid (Fig. 4A, lanes 3 and
4) (8). A block of GPI biosynthesis can also be observed
in vitro with YLL031c-depleted microsomes. WT microsomes
make the complete GPI precursor CP2 irrespective of the carbon source
on which cells have been grown (Fig. 4C, lanes 8 and 9). In contrast, YLL031c-depleted
microsomes make considerably less CP2 but instead accumulate a less
polar lipid that also has a higher RF than the M4
made by gpi7 microsomes (Fig. 4C,
lanes 3, 4, 10, and
11). This in vitro generated lipid comigrates with in vivo made 031b (Fig. 4C, lanes
2 and 4).
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Fig. 4.
YLL031c-depleted cells accumulate at least
two abnormal GPI lipids. A and B,
exponentially growing cells were radiolabeled with
[3H]Ins at 37 °C (4 µCi/1-2 × 107
cells), and desalted lipid extracts were analyzed by TLC using solvent
1 (A) or solvent 2 (B) and fluorography.
C, for lanes 1 and 2, cells
were labeled with [3H]Ins (I) and processed as
described for A. Lanes 3-14,
microsomes were prepared from exponentially growing cells and were
incubated with 3 or 6 µCi of UDP-[3H]GlcNAc
(GN), GDP-Man, tunicamycin, CoA, and ATP for 1 h at
30 °C (lanes 3-7) or 2 h at 30 °C
(lanes 8-14) as described (4), and lipid
extracts were analyzed by TLC using solvent 1 and fluorography.
Extracts in lanes 12 and 14 were first
treated with JBAM. FBY1102 and FBY1106 were grown in medium containing
either galactose (G) or glucose (D).
M(IP)2C,
mannosyldiinositolphosphorylceramide. The amounts of YLL031c activity
and Gpi7p, Mcd4p, and gpi10 activity are indicated as in Fig. 2.
gpi7 with YLL031c depletion strongly reduces
the accumulation of M4 and increases the accumulation of 031b (Fig. 4,
A, lane 6, and C,
lanes 5 and 6). Since the depletion of
YLL031c is only partial, it is understandable that in vitro there still is residual biosynthesis of CP2 or M4 in single or double
mutants, respectively (Fig. 4C, lanes
4, 6, and 11). The severely affected
mcd4-174 mutant has been reported to accumulate only trace
amounts of abnormal [3H]Ins-labeled lipids (10). We find
the same if Mcd4p is depleted using the repressible GAL1,10
promoter (Fig. 4A, lane 5).
-mannosidase, a linkage-specific exo--mannosidase cleaving 1-2 bonds, one obtains
Man2-GlcN-Ins (Fig. 5B). These data strongly
suggest that the bulk of lipid 031b has the structure
Man1-2Man1-2Man1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]Ins-PO4-lipid. We infer that the substituent on Man1 consists of EtN-P, because this substituent has been demonstrated to be present on Man1 of M2* of
the gpi10-1 mutant (4). The sensitivity of the in
vitro made lipid 031b to JBAM (Fig. 4C,
lanes 11 and 12) is in agreement with
the structure proposed above. A second, less prominent
Man2-GlcNAc-Ins peak in Fig. 5C could not be
reduced by increasing the concentration of JBAM. This suggests the
presence of a lipid having the structure Man1-2Man1-2[NH2-(CH2)2-PO4]Man1-6Man1-4GlcN1-6[acyl]Ins-PO4-lipid, which we call 031b' in the following. Based on its comigration with
031b on TLC we assume that 031b' also carries no more than one EtN-P
group.
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Fig. 5.
Analysis of the head group of lipid 031b
using HF, JBAM, and A. satoi
-mannosidase (ASAM). FBY1102
was labeled with [3H]Ins at 30 °C, and lipid 031b was
purified by two rounds of preparative TLC and used to prepare head
groups. Head groups were subjected to the following sequential
treatments. A, HF followed by N-acetylation;
B, HF followed by N-acetylation, desalting, and
then A. satoi -mannosidase; C, JBAM followed
by HF and then N-acetylation; D, HF followed by desalting,
then JBAM, and then N-acetylation. The thus generated
fragments were separated by paper chromatography, and radioactivity
contained in 1-cm-wide strips was determined through scintillation
counting. Standards 0-4 run on the same paper are
Manx-GlcNAc-Ins with x = 0, 1, 2, 4.
1-2Man1-2Man1-6Man1-4GlcN1-6[acyl]inositol-PO4-lipid. Thus, the GPI structures accumulating upon YLL031c depletion contain four mannoses and may contain HF-sensitive side chains on Man1 or Man2
but lack EtN-P on Man3 (Fig. 1).
View larger version (20K):
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Fig. 6.
Analysis of the head group of lipid 031a
using HF and JBAM. FBY1102 cells were labeled with
[3H]Ins at 30 °C, lipid 031a was purified by two
rounds of preparative TLC, and head groups were prepared. Head groups
were subjected to the following treatments. A, HF followed
by N-acetylation; B, JBAM followed by HF and then
N-acetylation; C, HF followed by desalting, JBAM,
and then N-acetylation. The thus generated fragments were
separated as in Fig. 5.
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Fig. 7.
Subcellular localization of YLL031c.
A, FBY1107 cell lysates were subjected to differential
centrifugations at 13,000 and 100,000 × g. These
centrifugations generated pellet P13, which contains ER, plasma, and
vacuolar membranes, and pellet P100, which contains Golgi membranes.
The 100,000 × g supernatant (S100) was
precipitated with trichloroacetic acid. B, FBY1107 cells
were converted to spheroplasts using various concentrations of
zymolyase-20T and then digested with proteinase K at the indicated
concentrations. Lane 6 shows the reactivity of
the anti-Myc antibody and secondary antibody with an extract from
FBY413 WT cells not harboring any Myc-tagged proteins. This control
extract was prepared as described in the legend to Fig. 3.
C, FBY1107 microsomes were sedimented at 13,000 × g for 15 min, and the membrane pellet was thoroughly
resuspended and digested with the indicated amounts of proteinase K at
0 °C on ice for 20 min in the presence or absence of 0.5% Triton
X-100. Samples were processed for SDS-polyacrylamide gel
electrophoresis and Western blotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Homology of YLL031c with GPI7
and MCD4. A, the first 395 amino
acids of YLL031c are shown. Potential N-glycosylation sites
and the N-terminal hydrophobic sequence for translocation of
the protein into the ER are underlined. Homologies with the
other S. cerevisiae (OSC) genes GPI7
and MCD4 were identified using the Clustal W (version
1.8) program at EBI. This introduced a few gaps into the YLL031c
sequence (dashes). In parallel, each of these three genes
was aligned with its nearest neighbors in other species, namely humans,
mice, C. elegans, and S. pombe.
GenBankTM accession numbers and Geninfo identifiers
of sequences aligned with YLL031c were T02245 GI:7513075, AAB93646
GI:2734088, and T40030 GI:7491546; aligned with GPI 7 were
Q09782 GI:1175452 and T21487 GI:7500059, and aligned with
MCD4 were NP_036459 GI:6912500, NP_038812 GI:7305383, and
T40715 GI:7491747. These alignments reveal homologies for the YLL031c
(C31) subfamily, the GPI7 (equivalent to YJL062w)
(C62) and the MCD4 (equivalent to YKL165c) (C65).
Asterisks, colons, and periods
indicate identity, strong similarity, and weak similarity,
respectively. B, in the Kyte-Doolittle plot of YLL031 are
integrated the positions of the nine potential
N-glycosylation sites (vertical
lines). The phosphatase motif (double
bar), and two motifs conserved between the YLL031c,
GPI7, and MCD4 subfamilies (single
bars) are also indicated.
YLL031c is homologous to GPI7 and MCD4 of S. cerevisiae, but closer homologues can be found in other species such as Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans or Schizosaccharomyces pombe. The GPI7/MCD4/YLL031c gene family can be subdivided into three subfamilies of more closely related genes. MCD4, GPI7, and YLL031c each belong to a different subfamily. All genes in this family predict proteins that have the same general structural attributes; i.e. they have an N-terminal signal sequence followed by a large hydrophilic domain and a C-terminal hydrophobic sequence containing numerous potential transmembrane domains. The hydrophilic domains of YLL031c, GPI7, and MCD4 have a distinct homology with mammalian enzymes classified as phosphodiesterases, phosphatases, or nucleotide pyrophosphatases. This homology extends over 240 amino acids. YLL031c and GPI7 also contain the distinct, universally conserved motif PTXTX8TGX2P (double bar above the sequence in Fig. 8A; stippled in Fig. 8B), which is found in mammalian but also some plant and bacterial enzymes. The homology among the members of the GPI7/MCD4/YLL031c gene family encompasses 400 amino acids at the N terminus of these proteins. The family contains several motifs that are not present or are only partially conserved in phosphatases (e.g. HXLGXDXXGH and DHGMXXXGXHG (single bar above the sequence in Fig. 8A; hatched in Fig. 8B). These motifs fall into regions in which also the highest degree of homology is observed within each subfamily. As shown in Fig. 8A, the degree of consensus within each subfamily is significantly higher than between YLL031c, GPI7, and MCD4 (compare lines OSC, C31, C62, and C65). Assuming that all of these genes evolved from a common ancestor, this suggests that the divergence and functional diversification of these three subfamilies occurred prior to the separation of the lineages leading to S. cerevisiae, S. pombe, C. elegans, and humans.
The phenotype of YLL031c-depleted cells strongly suggests that YLL031c indeed is involved in GPI anchoring. The cell wall fragility of YLL031c-depleted cells as revealed by CFW hypersensitivity is a common feature of all gpi mutants (33-36). Indeed, many GPI proteins of yeast are cell wall proteins or plasma membrane proteins participating in the building of the cell wall (37, 38). The appearance of abnormal GPI lipid intermediates and the specific accumulation or disappearance of mature and immature forms of GPI proteins equally point to a GPI anchoring defect.
This defect seems to be due to a defect in making GPI lipids but not in
the attachment process itself. This can be concluded, since
YLL031c-depleted microsomes still make some CP2 in vitro, but YLL031c-depleted cells do not accumulate CP2 in vivo as
is observed in mutants that are deficient in the GPI attachment process itself (6, 7). The difficulty of YLL031c-depleted cells seems to reside
in the attachment of the strategically important EtN-P to Man3, the
EtN-P that will have to make the link to the protein moiety (Fig. 1).
Earlier studies indicated that GPI7 is required for the
addition of an HF-sensitive substituent onto Man2, but not Man1 and
Man3, and that PIG-N, the human homologue of MCD4, is
required for the addition of EtN-P onto Man1 but not Man3. Together
with the data presented here, it therefore appears reasonable to
assume, as a working hypothesis, that MCD4, GPI7, and YLL031c are required for the addition of EtN-P to Man1, Man2, and
Man3, respectively. Moreover, their homology with phosphodiesterases suggests that these three genes encode the corresponding
EtN-P-transferases. (These transferases may be expected to bear
homology with phosphodiesterases, since previous studies by the group
of Anant Menon (39, 40) demonstrate that the attachment of the EtN-P to
Man3 occurs by transesterification of EtN-P from
phosphatidylethanolamine onto Man). A protein such as YLL031c
would seem to be well suited to carry out this transfer inasmuch as its
hydrophobic C-terminal domain may bind or even flip
phosphatidylethanolamine, and the hydrophilic phosphodiesterase domain
could operate the actual transfer reaction. Also, the subcellular
localization of YLL031c in the ER is compatible with its functioning as
an EtN-P-transferase, since it has been reported recently that, at
least up to the stage of
[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]Ins-PO4-lipid,
GPI lipids are made in the ER (41).
The structure of the abnormal GPI lipids accumulating in various
gpi mutant cells may not tell us in what order these
transfer reactions take place in WT cells, but, if we assume that the
observed GPI structures represent biosynthetic intermediates, they may tell us in what order the physiological pathway does not work. The six
theoretical possibilities for the order of EtN-P additions to Man1,
Man2, and Man3 starting from a Man4-GlcN-Ins core structure are represented in Fig. 9. Pathways
starting with reaction 1 (i.e. the addition of EtN-P by
YLL031c) may not be possible, since we then would expect that
MCD4-depleted cells or mcd4 mutants would accumulate the theoretical structure H1 or M4*, which is not the case
(Fig. 4A, lane 6) (10). Pathways using
reaction 3", leading from 031b to H2, may also be impracticable, since
YLL031c-depleted cells accumulate the substrate for reaction 3", but H2
is not observed. On the other hand, the appearance of 031b' in these cells suggests that reaction 3 is possible albeit not favored. Since
reactions 1 and 3" seem unfeasible and 3 is not favored, we may predict
that the physiological pathway in normal cells proceeds mainly via the
only remaining way, which is 2-1"-3
. Thus, the addition of EtN-P to
Man1 may represent a prerequisite for the addition of further EtN-P
residues. The fact that MCD4 is essential is in accordance
with a pivotal role of the EtN-P transfer to Man1. EtN-P is already
present on M2* of gpi10-1 having the structure
Man1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]Ins-PO4-lipid. Also, several studies in mammalian cells document that EtN-P may be
added before any other residues are attached to Man1 (11, 23, 42, 43).
Nevertheless, the abundance of 031a in YLL031c-depleted cells argues
that the addition of EtN-P to Man1 is not a prerequisite for the
addition of mannoses (Man2, Man3, and Man4, Fig. 1).
|
Since YLL031c is an essential gene, we may conclude that lipid 031b
cannot be used as an anchor device (i.e. that at least some
essential GPI proteins cannot be attached to the EtN-P on Man1 or that
this process, if it occurs, does not yield a functional GPI protein). A
similar conclusion has been reached previously in that the
gpi10-1 mutant does not seem to add the accumulating Man1-6[NH2-(CH2)2-PO4]Man1-4GlcN1-6[acyl]Ins-PO4-lipid
to proteins (4).
Genes other than YLL031c may be required for the transfer of EtN-P onto Man3. In mammals this step is dependent on PIG-F, a gene encoding a very hydrophobic protein. Its exact function has not yet been elaborated (44).
Further studies will be required to prove the validity of the model
proposed in Fig. 9 and to show that the MCD4/GPI7/YLL031c gene family indeed encodes EtN-P-transferases.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Drs Yves Bourbonnais, Hitoshi Shimoi, Dr. Y. Jigami, and Dr. M. Aebi for the generous gift of reagents. We thank Dr. Peter Orlean for sharing data before publication. We also thank Christine Vionnet and Anne Schneider for invaluable technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by Swiss National Foundation Grant 3100-032515.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.
To whom all 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@unifr.ch.
Published, JBC Papers in Press, May 22, 2000, DOI 10.1074/jbc.M003844200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
GPI, glycosylphosphatidylinositol;
CFW, Calcofluor White;
CP2, complete
precursor 2;
EtN-P, ethanolaminephosphate;
Ins, myo-inositol;
JBAM, jack bean -mannosidase;
nt, nucleotide(s);
SFH, short flanking homology;
WT, wild type;
PCR, polymerase chain reaction;
bp, base pair(s);
ER, endoplasmic
reticulum.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
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