cerevisiae genes implicated in addition of man-noses and EthN-P residues during GPI precursor assembly have been identified following characteriza-tion of the glycolipids that accumulate
Trang 1homologue are required for addition of the second
mannose during glycosylphosphatidylinositol precursor assembly
Anne-Lise Fabre1, Peter Orlean2and Christopher H Taron1
1 New England Biolabs, Beverly, MA, USA
2 Department of Microbiology, University of Illinois, Urbana, IL, USA
Glycosylphosphatidylinositols (GPIs) are key
glyco-lipids produced by all eukaryotes GPIs become
cova-lently attached to the C-termini of certain secretory
proteins and act as anchors to attach such proteins to
the outer face of the plasma membrane [1,2] Synthesis
of GPIs is essential for cell wall formation and
viabil-ity of yeast cells [3–5], for embryonic development in
mammalian cells [6], and for viability of the parasites
Leishmania mexicana [7] and the bloodstream form of
Trypanosoma brucei[8]
GPIs are assembled in the membranes of the
endo-plasmic reticulum (ER) by sequential addition of
components to phosphatidylinositol GPIs from all
organisms have a conserved core structure of NH2
-CH2-CH2-PO4
-6Mana1,2Mana1,6Mana1,4-GlcNa1,6-myo-inositol-PO4-lipid The three core mannoses may
be further modified with side-branching groups that vary between species For example, a fourth mannose (Man4) is side-branched to the third core mannose (Man3) of all yeast GPIs [9] and of certain human GPIs [10–12], and additional side-branching phospho-ethanolamines (EthN-Ps) may be added to the first and second mannoses of yeast [13–15] and mammalian GPIs [16,17]
S cerevisiae genes implicated in addition of man-noses and EthN-P residues during GPI precursor assembly have been identified following characteriza-tion of the glycolipids that accumulate in condicharacteriza-tional mutant strains The three a-linked mannoses compri-sing the GPI core are individually transferred from
Keywords
cell wall; glycosylphosphatidylinositol;
mannosyltransferase; Saccharomyces
cerevisiae
Correspondence
Christopher H Taron, New England Biolabs,
32 Tozer Road, Beverly, MA 01915, USA
Fax: +978 9211350
Tel: +978 9275054
E-mail: taron@neb.com
(Received 28 October 2004, revised 21
December 2004, accepted 4 January 2005)
doi:10.1111/j.1742-4658.2005.04551.x
Addition of the second mannose is the only obvious step in glycosylphos-phatidylinositol (GPI) precursor assembly for which a responsible gene has not been discovered A bioinformatics-based strategy identified the essential Saccharomyces cerevisiae Ybr004c protein as a candidate for the second GPI a-mannosyltransferase (GPI-MT-II) S cerevisiae cells depleted of Ybr004cp have weakened cell walls and abnormal morphology, are unable
to incorporate [3H]inositol into proteins, and accumulate a GPI intermedi-ate having a single mannose that is likely modified with ethanolamine phosphate These data indicate that Ybr004cp-depleted yeast cells are defective in second mannose addition to GPIs, and suggest that Ybr004cp
is GPI-MT-II or an essential subunit of that enzyme Ybr004cp homo-logues are encoded in all sequenced eukaryotic genomes, and are predicted
to have 8 transmembrane domains, but show no obvious resemblance to members of established glycosyltransferase families The human Ybr004cp homologue can substitute for its S cerevisiae counterpart in vivo
Abbreviations
CFW, Calcofluor white; Dol-P-Man, dolichol monophosphate mannose; ER, endoplasmic reticulum; EthN, ethanolamine; EthN-P,
ethanolamine phosphate; 5-FOA, 5-fluoro-orotic acid; GPI, glycosylphosphatidylinositol; GPI-MT, GPI a-mannosyltransferase; JbaM, jack bean a-mannosidase; Man1-GPI, mannosyl GPI; Man2-GPI, dimannosyl GPI; Man3-GPI, trimannosyl GPI; Man4-GPI, tetramannosyl GPI; PI-PLC, phospholipase C.
Trang 2Dol-P-Man [18] to GPI biosynthetic intermediates by
separate candidate GPI mannosyltransferases
(GPI-MT) The mammalian Pig-M protein and its yeast
ortholog Yjr013wp are required for addition of Man1
to GPI precursors [19], and the PIG-B⁄ Gpi10 proteins
for addition of Man3 [20–22] However, a candidate
GPI-MT-II has not yet been identified from any
organism and the transfer of Man2 to GPI glycans
remains the only obvious step of GPI precursor
syn-thesis or side-chain decoration for which a candidate
gene has not yet been discovered
We report here the identification of the yeast gene
encoding a novel 433 amino acid membrane protein
(Ybr004cp) required for addition of the second
man-nose to GPI precursors Yeast cells depleted of
Ybr004cp exhibit cell wall and morphological
abnor-malities, are defective in the incorporation of [3
H]ino-sitol into protein, and accumulate a GPI precursor
whose glycan contains a single mannose modified with
a substituent, probably EthN-P, that makes it
a-man-nosidase resistant Additionally, the human homologue
of Ybr004cp is able to substitute for its S cerevisiae
counterpart in vivo
Results
Identification of a candidate yeast GPI-MT-II
sequence
Although GPI MT-II might be expected to show
amino acid sequence similarity to known
Dol-P-Man-utilizing transferases such as GPI-MT-I, III, or IV, or
to protein: O-mannosyltransferases [23], searches of
protein sequence databases failed to identify any
sequences with statistically significant homology to the
above query sequences, suggesting that the yeast
GPI-MT-II has little resemblance to known
mannosyl-transferases at the primary sequence level
We therefore pursued an alternative,
bioinformatics-based strategy to identify candidate GPI MT-II
sequences We relied on a recent analysis of the
prote-ome of the pathogenic yeast Candida albicans in which
495 proteins with N-terminal signal sequences and that
likely localize to various compartments of the secretory
pathway were identified [24] We reasoned that this
subset of C albicans sequences likely included
GPI-MT-II We next eliminated sequences that failed to
meet the following criteria First, because GPI-MT I,
III, and IV are integral membrane proteins having at
least eight transmembrane domains and overall lengths
between 403 and 678 amino acids [25], we eliminated
sequences that had less than two predicted
transmem-brane domains or that had lengths greater than 1000
amino acids Second, we expected that the gene enco-ding GPI-MT-II would be essential, and we there-fore cross-referenced the remaining sequences to the
S cerevisiaeGenome Database (yeastgenome.org) keep-ing only sequences with obvious S cerevisiae homo-logues whose systematic gene deletions were lethal Third, we eliminated proteins with well-characterized functions, leaving only three sequences Finally, we expected GPI-MT-II to be encoded in every eukaryotic genome BLAST searches [26] against the GenBank database demonstrated that two of the three candidate proteins have homologues only in fungi, whereas the third, Ybr004cp, has homologues in fungi, mammals, plants, insects, nematodes and protozoa (Table 1) Thus, we considered Ybr004cp to be the most plaus-ible candidate S cerevisiae GPI-MT-II
Table 1 The Ybr004c protein sequence family % Identity ⁄ similar-ity calculated relative to S cerevisiae sequence.
Organism
Length (amino acids)
% Identity ⁄ similarity
GenBank accession number Fungi
Cryptosporidium parvum 436 23 ⁄ 71 CAD98327
Encephalitozoon cuniculi 393 20 ⁄ 60 NP_596980 Eremothecium gossypii 427 47 ⁄ 77 NP_984865
Schizosaccharomyces pombe 426 24 ⁄ 60 NP_592878
Mammals
Plants
Insects Drosophila melanogaster 449 17 ⁄ 51 AAF23239 Fish
Tetraodon nigroviridis 494 22 ⁄ 56 CAG00037 Nematodes
Caenorhabditis briggsae 673 20 ⁄ 59 CAE67131 Caenorhabditis elegans 672 19 ⁄ 61 NP_491783 Protozoa
Plasmodium falciparum 503 16 ⁄ 53 NP_701814
Trang 3Growth and GPI anchoring defects of
Ybr004cp-depleted strains
To establish whether Ybr004cp is involved in GPI
assembly, we tested whether depletion of this protein in
YBR004c-disrupted haploid cells leads to a GPI assembly
defect We constructed a YBR004c-disrupted haploid
strain in which expression of a plasmid-borne wild-type
allele of YBR004c is regulated by the glucose-repressible
GAL10 promoter (ybr004cD-pGAL-YBR004c) When
grown in medium containing glucose, expression of
YBR004cis repressed, uncovering recessive phenotypes
associated with depleting cells of Ybr004cp We tested
this strain for growth and biochemical defects
charac-teristic of a GPI anchoring deficiency
Strains defective in GPI anchoring are typically
hypersensitive to the fluorescent dye Calcofluor white
(CFW) and have weakened cell walls [27] This was
the case for ybr004cD-pGAL-YBR004c cells which
showed impaired growth compared to a wild-type
strain on medium containing glucose and 16 lg CFW
per mL (Fig 1A) Furthermore, glucose-grown
ybr004cD-pGAL-YBR004c cells examined by
phase-contrast microscopy were generally large, misshapen,
and clumpy (Fig 1B), phenotypes indicating a loss of
cell wall integrity and seen with other gpi mutants [28]
Because GPI-anchored proteins are the only known
proteins covalently linked to inositol in yeast [29,30], we
examined the ability of Ybr004cp-depleted cells to
incor-porate [3H]inositol into proteins The
ybr004cD-pGAL-YBR004cstrain was grown and labeled with [3H]inositol
in medium containing galactose or glucose to promote
or repress YBR004c expression, respectively
Radiolabe-led cells were lysed in detergent and extracted proteins
were separated by SDS⁄ PAGE, after which [3
H]inositol-labeled proteins were detected by fluorography
Wild-type cells were capable of forming [3H]inositol-labeled
GPI anchored proteins in medium containing either
galac-tose or glucose (Fig 1C, lanes 1, 2), whereas
ybr004cD-pGAL-YBR004c cells incorporated significantly less
[3H]inositol into proteins in glucose-containing medium
(Fig 1C, lane 4), where YBR004c expression is
repressed Thus, Ybr004cp-depleted cells exhibit a
global defect in formation of GPI-anchored proteins
Ybr004cp-depleted cells accumulate a novel
GPI precursor
Yeast strains with conditional defects in mannosylation
and EthN-P addition to the GPI precursor or in GPI
transfer to protein accumulate GPI assembly
inter-mediates that can be detected by pulse-radiolabeling
such strains under nonpermissive conditions [14,15,
31,32] The step in GPI assembly affected in such mutants can be inferred from the structure of the accu-mulating GPI Therefore, we looked for evidence
of lipid accumulation in glucose-repressed ybr004cD-pGAL-YBR004c cells The strain was metabolically labeled with [3H]inositol, after which lipids were extracted from cells, separated by TLC, and [3 H]inosi-tol-labeled lipids were detected by fluorography Cells radiolabeled under repressing conditions accumulated
an aberrant [3H]inositol-containing lipid (lipid 004–1; Fig 2A, lane 4) that was nearly absent from lipids iso-lated from cells grown in medium containing galactose (Fig 2A, lane 3) Lipid 004–1 was susceptible to treat-ment with mild-base (Fig 2B, lane 2) and resistant to cleavage by PI-PLC (Fig 2B, lane 4), indicating that it contained ester-linked fatty acids and an inositol acyl chain, respectively This combination of traits is a characteristic of lipid intermediates in GPI precursor synthesis Finally, lipid 004–1 migrated as a less polar species than the previously characterized Man2- and Man3-GPIs that accumulate in cells defective in
A
Fig 1 ybr004c mutants have defects in cell wall synthesis, mor-phogenesis, and GPI anchoring (A) Ten-fold serial dilutions of wild-type (wt) or ybr004cD-pGAL-YBR004c cells were spotted onto YPD agar-containing medium with or without 16 lg CFW per mL and grown 3 days at 30 C (B) ybr004cD-pGAL-YBR004c cells were grown either in galactose- (Gal) or glucose-containing (Glc) medium Cellular phenotypes were observed by phase contrast microscopy (C) Proteins from wt and ybr004cD-pGAL-YBR004c strains were metabolically labeled with [3H]inositol in medium containing either galactose or glucose for 60 min at 30 C Proteins were extracted from cells, separated by SDS ⁄ PAGE and radiolabeled GPI anchored proteins were visualized by fluorography.
Trang 4addition of the third [21,22] and fourth [31] mannoses
to GPI precursors (Fig 3B, and data not shown), sug-gesting that it is a GPI intermediate that forms prior
to addition of Man3 and -4 to yeast GPI precursors
A yeast strain defective in GPI-MT-II would be pre-dicted to accumulate a GPI intermediate bearing a sin-gle mannose that may or may not be substituted with
a side-branching EthN-P residue Phosphatidylethanol-amine, the donor of EthN-P residues to Man1 and -3
of GPIs [33,34], can be synthesized either de novo from exogenous ethanolamine (EthN), or by decarboxylation
of phosphatidylserine Metabolic labeling experiments using [14C]EthN or [3H]serine were therefore carried out to determine if lipid 004–1 contains an EthN-P moi-ety To enhance [14C]EthN incorporation into lipids, radiolabeling was carried out in a ybr004cD-pGAL-YBR004c⁄ psd1D ⁄ psd2D strain, which lacks phosphati-dylserine decarboxylase activity (see Experimental procedures) This strain accumulated lipid 004–1 upon labeling with [14C]EthN in medium containing glucose (Fig 2C, lane 3), but not in galactose-containing medium (Fig 2C, lane 2) Similarly, ybr004cD-pGAL-YBR004c cells accumulated lipid 004–1 upon labeling with [3H]serine in the presence of glucose (Fig 2C, lane 5) Taken together, these results are strong evidence that lipid 004–1 contains EthN-P, and therefore that 004–1 contains at least one mannose residue
We next compared the TLC mobility of lipid 004–1
to that of a Man1(EthN-P)-GPI mobility standard derived from the previously characterized GPI interme-diate that accumulates upon depletion of Gpi13p, the GPI EthN-P transferase that adds EthN-P to Man3 [14,15] The GPI that accumulates in gpi13D-pGAL-GPI13 cells is a Man4-GPI, much of which is modified
by a single EthN-P on Man1, but lesser amounts of which bear their EthN-P on Man2 [15] Treatment of the major Man4-GPI isoform with JbaM would there-fore yield a GPI with a single mannose bearing
EthN-P [a Man1(EthN-P)-GPI], whereas the minor isoform would be converted to a Man2(EthN-P)Man1-GPI The Man1(EthN-P)-GPI comigrated with lipid 004–1
on TLC (Fig 2D, lanes 1 and 4) suggesting the two share the same structure A GPI precursor with the thin layer chromatographic mobility of Man1 (EthN-P)-GPI has not previously been reported to accumulate in any yeast GPI assembly mutant In addition, lipid 004–1 was resistant to treatment with JbaM, indicating that it lacks an unsubstituted terminal mannose (Fig 2D, lane 2) JbaM treatment of lipids from Ybr004cp-depleted cells also generated some very non-polar material whose mobility is consistent with that
of GlcN [acyl-Ins]PI, which may have originated from
an unsubstituted Man1-GPI that may comigrate with
Fig 2 ybr004cD-pGAL-YBR004c cells accumulate a putative Man 1
-(EthN-P)-GPI (A) Wild-type and ybr004cD-pGAL-YBR004c cells
were grown and [ 3 H]inositol-labeled in galactose- (lanes 1 and 3) or
glucose-containing medium (lanes 2 and 4) to induce or repress
YBR004c expression, respectively Extracted lipids were separated
by TLC (B) ybr004cD-pGAL-YBR004c cells were grown and [ 3
H]ino-sitol-labeled in glucose-containing medium Lipids were extracted
from cells and incubated either with or without mild-base (lanes 1
and 2) and with or without PI-PLC (lanes 3 and 4) (C) Lipids were
extracted from [ 3 H]inositol- (lane 1) or [ 3 H]serine-labeled (lanes 4
and 5) ybr004cD-pGAL-YBR004c cells or from [ 14 C]EthN-labeled
ybr004cD-pGAL-YBR004c ⁄ psd1D ⁄ psd2D cells (lanes 2 and 3) and
separated by TLC Lane 1 is from a 3-day film exposure that was
digitally cropped and precisely re-aligned with adjacent lanes 2–5,
which were exposed to film for 10 days (D) Lipids were extracted
from ybr004cD-pGAL-YBR004c or gpi13D-pGAL-GPI13 cells grown
and [3H]inositol labeled in glucose-containing medium and
incuba-ted with or without JbaM (lanes 1–4) prior to their separation by
TLC The lipid that accumulates in gpi13D-pGAL-GPI13 cells is a
mixture of two Man 4 -GPI isoforms that each bear a single EthN-P
on either Man1 or Man2 [15] JbaM treatment digests Man4-GPI
(lane 3) into a Man2(EthN-P)Man1-GPI and a Man1
(EthN-P)-GPI (lane 4) Lipid 004–1 (lanes 1 and 2) comigrates with the
Man 1 (EthN-P)-GPI (lane 4) M1, M2 and M3 represent GPI
man-noses in the order of their addition to GPIs; PE,
phosphoethanol-amine; G, glucosphosphoethanol-amine; PI, phosphatidylinositol.
Trang 5[3H]inositol-labeled non-GPIs in this chromatographic
solvent system, obscuring its detection
Taken together, these data strongly suggest that lipid
004–1 is a GPI intermediate containing a single mannose
substituted with a side-branching EthN-P residue, and
corresponds to GPI species H5 in mammalian cells [35],
which can be generated by JbaM treatment of
mamma-lian Man3(EthN-P)-GPI [36] The accumulation of this
GPI suggests that ybr004cD-pGAL-YBR004c cells have
a defect in addition of Man2 to GPI precursors
Epistasis tests place Ybr004cp in the GPI
biosynthetic pathway
To obtain genetic evidence that YBR004c functions in
the GPI biosynthetic pathway, the epistasis
relation-ships to genes upstream and downstream of Man2
addition to GPIs were tested Two double mutant
strains were created by mating haploids harboring
either smp3–2 or Dgpi1 temperature-sensitive alleles
with the ybr004cD-pGAL-YBR004c strain and the
[3H]inositol-labeled lipids they accumulate at 37C
under repressing conditions were examined
At 37C, the Dgpi1 mutation, which blocks the
transfer of GlcNAc to phosphatidylinositol (PI),
the first step of GPI precursor assembly [28], blocks
the accumulation of lipid 004–1 gpi1D ⁄
ybr004cD-pGAL-YBR004c cells grown and labeled at 25C in
medium containing glucose showed prominent
accu-mulation of lipid 004–1 (Fig 3A, lane 5) However,
the same cells grown in glucose-containing medium
at 37C showed no accumulation of lipid 004–1
(Fig 3A, lane 6) indicating that formation of 004–1 is dependent upon GlcNAc-PI synthesis
An analogous experiment was performed with an smp3–2⁄ ybr004cD-pGAL-YBR004c double mutant smp3–2 mutants are defective in addition of Man4 to GPI precursors and accumulate a Man3-GPI inter-mediate [31] smp3–2⁄ ybr004cD-pGAL-YBR004c cells grown and [3H]inositol-labeled in medium containing galactose prominently accumulate the Man3-GPI at
25C (Fig 3B, lane 3) and to a lesser degree at 37 C (Fig 3B, lane 4) However, lipids from double mutant cells labeled in glucose medium at 25C contain predominantly lipid 004–1 and significantly less Man3 -GPI (Fig 3B, lane 5), indicating that Ybr004cp func-tions upstream of Smp3p Together, these data further support the conclusion that Ybr004cp functions in the yeast GPI assembly pathway
Sequence analysis of the Ybr004cp protein family Database searches using the S cerevisiae Ybr004cp protein sequence and the Psi-BLAST algorithm revealed 25 similar sequences in various eukaryotes, including Homo sapiens (Table 1) No significant homology was observed between Ybr004cp and proteins from prokaryotes, and no eukaryotic genome encoded obvious additional Ybr004cp-like sequences The consensus membrane topology predictive algorithm of Persson and Argos [37] suggests that Ybr004c proteins typically have eight transmembrane domains with four intraluminally oriented loops (Fig 4) Alignment of all members of the Ybr004cp
Fig 3 ybr004c acts downstream of gpi1 and upstream of smp3 in the GPI biosyn-thetic pathway (A) A gpi1D ⁄ ybr004cD-pGAL-YBR004c double mutant strain was radiolabeled with [3H]inositol in SGalYE medium at 25 C or 37 C (lanes 3 and 4),
or in SGlcYE at 25 C or 37 C (lanes 5 and 6) Lipids were extracted from cells and sep-arated by TLC Lipid 004-1 accumulates in glucose-containing medium at 25 C (lane 5) but does not when the temperature-sensi-tive gpi1 allele is suppressed at 37 C (lane 6) (B) An smp3–2 ⁄ ybr004cD-pGAL-YBR004c double mutant strain was [3H]inositol-labeled
as described above after which lipids were extracted and separated by TLC.
Trang 6family (Supplementary Fig S1) revealed three invariably
conserved residues (Glu, Gln, and Trp) that each are
predicted to reside within an intraluminal loop (Fig 4)
Expression of human YBR004c restores viability
to Dybr004c yeast
We tested if the human Ybr004c homologue (GenBank
NP_060307) could complement the lethal ybr004c::
KanR null mutation in vivo in S cerevisiae
Heterozy-gous ybr004c::KanR⁄ YBR004c ura3 ⁄ ura3 diploids were
transformed with pGAL-hYBR004c Transformants
were sporulated and asci were dissected onto YPGal
agar medium to assess the viability of the individual
haploid spores Asci from diploids harboring
pGAL-hYBR004c gave rise to four viable haploid progeny
Additionally, two haploids from each tetrad were
resistant to G418 (Fig 5A) and sensitive to 5-FOA
(Fig 5B), indicating that they harbored the ybr004c::
KanRallele and that their viability was dependent upon
the complementing URA3-containing plasmid
Addition-ally, neither pGAL-hYBR004c nor pGAL-YBR004c
were able to complement lethal null mutations of
YJR013w, GPI10, or SMP3, genes encoding the
mannosyltransferases that add Man1 [19], Man3 [22]
and Man4 [31] to yeast GPI precursors, respectively
Therefore, hYBR004c expression specifically restores
viability to yeast defective in Man2 addition to GPIs
We conclude that human Ybr004cp is the functional
equivalent of S cerevisiae Ybr004cp
Discussion
The majority of the steps in assembly and decoration
of the GPI precursor glycolipid have been defined
genetically in that at least one gene’s product has been implicated in all but one of the predicted reactions in the GPI pathway The exception is the addition of the second mannose to the GPI core We show here that depletion of the essential, multispanning membrane protein Ybr004cp from yeast cells leads to the bio-chemical defects expected if addition of the second, a-1,6-linked mannose to GPI precursors is prevented These defects are a block in the incorporation of [3H]inositol into protein, consistent with abolition of GPI anchoring, and the accumulation of a PI-PLC-resistant, base-labile [3H]inositol-labeled glycolipid whose glycan headgroup likely contains a single man-nose that is modified with an EthN-P residue
Our epistasis tests with known GPI assembly mutants indicate that Ybr004cp functions in the GPI assembly pathway, and further, Ybr004cp-depletion gives rise to cell wall and morphological defects charac-teristic of GPI assembly mutants We therefore propose that Ybr004cp is an excellent candidate for GPI-MT-II itself or an essential subunit of that enzyme
Our results also shed light on the first EthN-P addi-tion step in yeast Because the GPI precursor that accumulates when addition of Man2 is blocked is modified with phosphoethanolamine, EthN-P can be
Fig 4 Predicted membrane topology of Ybr004c proteins The
fig-ure was drawn using data predicted by alignment of 25 Ybr004c
protein sequences (Fig S1) using the CLUSTAL W program [42]
fol-lowed by analysis of the aligned sequences using the TMAP
algo-rithm [37] to predict conserved membrane topology as described in
Experimental procedures Black circles represent the position of
strictly conserved amino acids, whereas gray circles indicate amino
acids conserved in > 85% (22 of 25) of the aligned sequences
Pre-dicted loop lengths range from the shortest to the longest size
observed in all 25 sequences.
A
B
Fig 5 Human YBR004c expression restores viability to Dybr004c
S cerevisiae cells A heterozygous ybr004c::Kan R ⁄ YBR004c diploid yeast strain harboring the pGAL-hYBR004c expression vector was sporulated and tetrads microdissected onto YPGal agar medium For tetrads giving rise to four viable progeny, each haploid segre-gant was streaked on YPGal agar medium containing either 200 lg G418 per mL (A) or 1 mg 5-FOA per mL (B) and grown for 3 days
at 25 C.
Trang 7added to Man1 of GPI precursors as early as the
Man1-GPI stage
To date, no biochemical function has been described
for any Ybr004c protein, although its Drosophila
homo-logue (termed ‘vegetable’) was identified in a screen
for genes implicated in formation of the peripheral
ner-vous system [38] These findings, and our assignment of
function to Ybr004c proteins, suggest the importance of
efficient GPI anchoring in this developmental process
Our identification of a novel, conserved protein
essential for Man2 addition to GPIs will allow us to
carry out detailed biochemical and genetic analyses of
this uncharacterized step in GPI biosynthesis
Experimental procedures
Materials
[2-3H]-myo-Inositol (sp act 30 CiÆmmol )1), [1,2–14
C]-etha-nolamine hydrocloride and L-[3H(G)]-serine were obtained
from American Radiolabeled Chemicals Calcofluor white
(fluorescent brightener 28), Geneticin (G418), Jack bean
a-mannosidase (JbaM), phospholipase C (PI-PLC) and
5-fluoroorotic acid (5-FOA) were from Sigma
Yeast strains and media
SD (SGlc) and YPD media were made as described [39]
YPGal medium has the same composition as YPD but with
2% (w⁄ v) galactose instead of glucose Inositol-free
syn-thetic medium and synsyn-thetic medium containing 0.2% yeast
extract (w⁄ v) and glycerol (SGlyYE), galactose (SGalYE)
or glucose (SGlcYE) were prepared as described [15]
Cal-cofluor white hypersensitivity was tested on YPD agar
con-taining 16 lg Calcofluor white per mL Sensitivity of yeast
to 5-FOA was determined on YPGal medium containing
1 mg 5-FOA per mL
Diploid heterozygous YBR004c⁄ ybr004c::KanR,
YJR013-w⁄ yjr013w::KanR, GPI10⁄ gpi10::KanR and SMP3⁄ smp3::
KanR strains were purchased from Research Genetics To
construct a glucose-repressible allele of YBR004c, the
YBR004c⁄ ybr004c::KanR
heterozygous diploid was trans-formed with pGAL-YBR004c (see below) Transformants
were sporulated and tetrads dissected Haploid progeny
harboring a ybr004c::KanR allele complemented by
pGAL-YBR004c were identified by growth on YPGal plates
con-taining 200 lg G418 per mL The double mutant strains
gpi1D ⁄ ybr004cD-pGAL-YBR004c and smp3–2⁄
YBR004c were created by mating
ybr004cD-pGAL-YBR004c (MAT a, his3D1, leu2D1, ura3D0, met15D0,
ybr004c::KanR) with Dgpi1 [28] and smp3–2 [31] strains,
respectively A ybr004cD-pGAL-YBR004c strain
back-ground harboring an ethanolamine auxotrophy was created
by mating ybr004cD-pGAL-YBR004c with RYY51 (MAT
a, trp1–1, ura3–1, leu2–3,112, his3–11, suc2, rho+, lys2, psd1::TRP1, psd2::HIS3) [40]
Construction of YBR004c yeast expression plasmids
The human (GenBank NP_060307) and S cerevisiae YBR004c genes were PCR-amplified from human liver cDNA or S cerevisiae genomic DNA, respectively Each was cloned as a EcoRI-BamHI fragment downstream of the galactose-inducible⁄ glucose-repressible GAL10-1 promoter
in vector pMW20 [41] to produce the pGAL-hYBR004c (human) and pGAL-YBR004c (yeast) S cerevisiae expres-sion plasmids
In vivo radiolabeling of S cerevisiae lipids and thin layer chromatography
[3H]Inositol labeling of lipids in temperature-sensitive yeast strains was performed as previously described [15] For [3H]inositol or [3H]serine labeling of the ybr004cD-pGAL-YBR004cstrain, cells were first grown in SGlyYE medium, then shifted to SGlcYE or SGalYE medium for 16 h and labeled for 2 h at 30C with 15 lCi [3
H]inositol or 50 lCi [3H]serine [14C]Ethanolamine labeling of theDpsd1 ⁄ Dpsd2 ⁄ ybr004cD-pGAL-YBR004c strain was performed in the same manner except that each growth medium was supplemented with 5 mm ethanolamine and 5 mm choline, and metabolic labeling was performed with 20 lCi [14C]ethanolamine for
23 h at 25 C For radiolabeling of double mutant strains,
cells were grown in SGlyYE medium for 2 days at 25C, then grown in SGalYE or SGlcYE medium for 16 h Cells were shifted to 25C or 37 C for 20 min and radiolabeled with 15 lCi [3H]inositol for 2 h Radiolabeled lipids were extracted from cells and treated with mild-base, phospho-lipase C, or JbaM as described [12,15]
Isolated lipids were separated by TLC on silica 60 plates (VWR) TLC plates were prerun in chloroform⁄ meth-anol⁄ water (65 : 25 : 4, v ⁄ v ⁄ v), after which lipids were applied and separated in chloroform–methanol–water (5 : 5 : 1, v⁄ v ⁄ v) TLC-separated lipids were exposed to BioMax MS film (Eastman Kodak) for 1–4 days using a BioMax Transcreen LE intensifier screen
[3H]Inositol labeling of proteins in ybr004cD-pGAL-YBR004c cells was performed as described [15] [3 H]ino-sitol-labeled proteins were separated on a 10–20% SDS⁄ PAGE (Daichii) and detected by fluorography as des-cribed above
Protein sequence analysis Consensus topology prediction for 25 Ybr004c proteins (Table 1 and supplementary Fig S1) was performed using the program clustal w [42] to align the primary amino
Trang 8acid sequences (parameters: protein weight matrix,
BLO-SUM series; gap open penalty, 10; gap extension penalty,
0.1) The aligned sequences were submitted as input to the
tmap program [37] to predict conserved membrane
topol-ogy using default parameters
Acknowledgements
CHT thanks Dr Donald Comb of New England
Bio-labs for financial support PO is supported by National
Institutes of Health Grant GM46220 The authors
thank B Taron and P Colussi for advice and technical
assistance
References
1 McConville MJ & Ferguson MA (1993) The structure,
biosynthesis and function of glycosylated
phosphatidyl-inositols in the parasitic protozoa and higher
eukar-yotes Biochem J 294, 305–324
2 Tiede A, Bastisch I, Schubert J, Orlean P & Schmidt
RE (1999) Biosynthesis of glycosylphosphatidylinositols
in mammals and unicellular microbes Biol Chem 380,
503–523
3 Leidich SD, Drapp DA & Orlean P (1994) A
condition-ally lethal yeast mutant blocked at the first step in
gly-cosyl phosphatidylinositol anchor synthesis J Biol
Chem 269, 10193–10196
4 Colussi PA & Orlean P (1997) The essential
Schizosac-charomyces pombegpil+gene complements a bakers’
yeast GPI anchoring mutant and is required for efficient
cell separation Yeast 13, 139–150
5 Grimme SJ, Colussi PA, Taron CH & Orlean P (2004)
Deficiencies in the essential Smp3 mannosyltransferase
block glycosylphosphatidylinositol assembly and lead to
defects in growth and cell wall biogenesis in Candida
albicans Microbiology 150, 3115–3128
6 Kawagoe K, Kitamura D, Okabe M, Taniuchi I, Ikawa
M, Watanabe T, Kinoshita T & Takeda J (1996)
Glyco-sylphosphatidylinositol-anchor-deficient mice:
implica-tions for clonal dominance of mutant cells in
paroxysmal nocturnal hemoglobinuria Blood 87, 3600–
3606
7 Ilgoutz SC, Zawadzki JL, Ralton JE & McConville MJ
(1999) Evidence that free GPI glycolipids are essential for
growth of Leishmania mexicana EMBO J 18, 2746–2755
8 Nagamune K, Nozaki T, Maeda Y, Ohishi K, Fukuma
T, Hara T, Schwarz RT, Sutterlin C, Brun R, Riezman
H & Kinoshita T (2000) Critical roles of
glycosylpho-sphatidylinositol for Trypanosoma brucei Proc Natl
Acad Sci USA 97, 10336–10341
9 Fankhauser C, Homans SW, Thomas-Oates JE,
McConville MJ, Desponds C, Conzelmann A &
Fergu-son MA (1993) Structures of
glycosylphosphatidylinosi-tol membrane anchors from Saccharomyces cerevisiae
J Biol Chem 268, 26365–26374
10 Homans SW, Ferguson MA, Dwek RA, Rademacher
TW, Anand R & Williams AF (1988) Complete struc-ture of the glycosyl phosphatidylinositol membrane anchor of rat brain Thy-1 glycoprotein Nature 333, 269–272
11 Roberts WL, Santikarn S, Reinhold VN &
Rosenberry TL (1988) Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry J Biol Chem 263, 18776–18784
12 Taron BW, Colussi PA, Wiedman JM, Orlean P & Taron CH (2004) Human Smp3p adds a fourth man-nose to yeast and human glycosylphosphatidylinositol precursors in vivo J Biol Chem 279, 36083–36092
13 Benachour A, Sipos G, Flury I, Reggiori F, Canivenc-Gansel E, Vionnet C, Conzelmann A & Benghezal M (1999) Deletion of GPI7, a yeast gene required for addi-tion of a side chain to the glycosylphosphatidylinositol (GPI) core structure, affects GPI protein transport, remodeling, and cell wall integrity J Biol Chem 274, 15251–15261
14 Flury I, Benachour A & Conzelmann A (2000) YLL031cbelongs to a novel family of membrane pro-teins involved in the transfer of ethanolaminephosphate onto the core structure of glycosylphosphatidylinositol anchors in yeast J Biol Chem 275, 24458–24465
15 Taron CH, Wiedman JM, Grimme SJ & Orlean P (2000) Glycosylphosphatidylinositol biosynthesis defects
in Gpi11p- and Gpi13p-deficient yeast suggest a branched pathway and implicate gpi13p in phos-phoethanolamine transfer to the third mannose Mol Biol Cell 11, 1611–1630
16 Ueda E, Sevlever D, Prince GM, Rosenberry TL, Hirose S & Medof ME (1993) A candidate mammalian glycoinositol phospholipid precursor containing three phosphoethanolamines J Biol Chem 268, 9998–10002
17 Hong Y, Maeda Y, Watanabe R, Ohishi K, Mishkind
M, Riezman H & Kinoshita T (1999) Pig-n, a mamma-lian homologue of yeast Mcd4p, is involved in transfer-ring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol J Biol Chem 274, 35099– 35106
18 Menon AK, Mayor S & Schwarz RT (1990) Biosynth-esis of glycosyl-phosphatidylinositol lipids in Trypano-soma brucei: involvement of
mannosyl-phosphoryldolichol as the mannose donor EMBO J 9, 4249–4258
19 Maeda Y, Watanabe R, Harris CL, Hong Y, Ohishi K, Kinoshita K & Kinoshita T (2001) PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER EMBO J 20, 250–261
Trang 920 Takahashi M, Inoue N, Ohishi K, Maeda Y, Nakamura
N, Endo Y, Fujita T, Takeda J & Kinoshita T (1996)
PIG-B, a membrane protein of the endoplasmic
reticu-lum with a large reticu-lumenal domain, is involved in
trans-ferring the third mannose of the GPI anchor EMBO J
15, 4254–4261
21 Canivenc-Gansel E, Imhof I, Reggiori F, Burda P,
Con-zelmann A & Benachour A (1998) GPI anchor
bio-synthesis in yeast: phosphoethanolamine is attached to
the alpha1,4-linked mannose of the complete precursor
glycophospholipid Glycobiology 8, 761–770
22 Su¨tterlin C, Escribano MV, Gerold P, Maeda Y, Mazon
MJ, Kinoshita T, Schwarz RT & Riezman H (1998)
Saccharomyces cerevisiae GPI10, the functional
homolo-gue of human PIG-B, is required for
glycosylphosphati-dylinositol-anchor synthesis Biochem J 332, 153–159
23 Strahl-Bolsinger S, Gentzsch M & Tanner W (1999)
Protein O-mannosylation Biochim Biophys Acta 1426,
297–307
24 Lee SA, Wormsley S, Kamoun S, Lee AF, Joiner K &
Wong B (2003) An analysis of the Candida albicans
gen-ome database for soluble secreted proteins using
compu-ter-based prediction algorithms Yeast 20, 595–610
25 Oriol R, Martinez-Duncker I, Chantret I, Mollicone R
& Codogno P (2002) Common origin and evolution of
glycosyltransferases using Dol-P-monosaccharides as
donor substrate Mol Biol Evol 19, 1451–1463
26 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang
Z, Miller W & Lipman DJ (1997) Gapped blast and
PSI- blast: a new generation of protein database search
programs Nucleic Acids Res 25, 3389–3402
27 Ram AF, Wolters A, Ten Hoopen R & Klis FM (1994)
A new approach for isolating cell wall mutants in
Sac-charomyces cerevisiaeby screening for hypersensitivity
to calcofluor white Yeast 10, 1019–1030
28 Leidich SD & Orlean P (1996) Gpi1, a Saccharomyces
cerevisiaeprotein that participates in the first step in
glycosylphosphatidylinositol anchor synthesis J Biol
Chem 271, 27829–27837
29 Conzelmann A, Fankhauser C & Desponds C (1990)
Myoinositol gets incorporated into numerous membrane
glycoproteins of Saccharomyces cerevisiae; incorporation
is dependent on phosphomannomutase (Sec53) EMBO
J 9, 653–661
30 Orlean P (1990) Dolichol phosphate mannose synthase
is required in vivo for glycosyl phosphatidylinositol
membrane anchoring, O mannosylation, and N
glycosy-lation of protein in Saccharomyces cerevisiae Mol Cell
Biol 10, 5796–5805
31 Grimme SJ, Westfall BA, Wiedman JM, Taron CH &
Orlean P (2001) The essential Smp3 protein is required
for addition of the side-branching fourth mannose
during assembly of yeast glycosylphosphatidylinositols
J Biol Chem 276, 27731–27739
32 Benghezal M, Lipke PN & Conzelmann A (1995) Iden-tification of six complementation classes involved in the biosynthesis of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae J Cell Biol 130, 1333–1344
33 Menon AK & Stevens VL (1992) Phosphatidylethanol-amine is the donor of the ethanolPhosphatidylethanol-amine residue linking
a glycosylphosphatidylinositol anchor to protein J Biol Chem 267, 15277–15280
34 Imhof I, Canivenc-Gansel E, Meyer U & Conzelmann
A (2000) Phosphatidylethanolamine is the donor of the phosphorylethanolamine linked to the a1,4-linked man-nose of yeast GPI structures Glycobiology 10, 1271– 1275
35 Hirose S, Prince GM, Sevlever D, Ravi L, Rosenberry
TL, Ueda E & Medof ME (1992) Characterization of putative glycoinositol phospholipid anchor precursors in mammalian cells: localization of phosphoethanolamine
J Biol Chem 267, 16968–16974
36 Hong Y, Maeda Y, Watanabe R, Inoue N, Ohishi K & Kinoshita T (2000) Requirement of PIG-F and PIG-O for transferring phosphoethanolamine to the third man-nose in glycosylphosphatidylinositol J Biol Chem 275, 20911–20919
37 Persson B & Argos P (1994) Prediction of transmem-brane segments in proteins utilising multiple sequence alignments J Mol Biol 237, 182–192
38 Prokopenko SN, He Y, Lu Y & Bellen HJ (2000) Muta-tions affecting the development of the peripheral ner-vous system in Drosophila: a molecular screen for novel proteins Genetics 156, 1691–1715
39 Sherman F (1991) Getting started with yeast Methods Enzymol 194, 3–21
40 Trotter PJ & Voelker DR (1995) Identification of a non-mitochondrial phosphatidylserine decarboxylase activity (PSD2) in the yeast Saccharomyces cerevisiae
J Biol Chem 270, 6062–6070
41 Zieler HA, Walberg M & Berg P (1995) Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein Mol Cell Biol 15, 3227– 3237
42 Thompson JD, Higgins DG & Gibson TJ (1994) clustal w: improving the sensitivity of progressive mul-tiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673–4680
Supplementary material
The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4551/EJB4551sm.htm
Fig S1 Multiple sequence alignment of 25 Ybr004c proteins