Saccharomyces cerevisiae Ybr004c and its human homologue are required for addition of the second mannose during glycosylphosphatidylinositol precursor assembly Anne-Lise Fabre 1 , Peter Orlean 2 and Christopher H. Taron 1 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 NH 2 - CH 2 -CH 2 -PO 4 -6Mana1,2Mana1,6Mana1,4-GlcNa1,6- myo-inositol-PO 4 -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 conditional 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 [ 3 H]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; Man 1 -GPI, mannosyl GPI; Man 2 -GPI, dimannosyl GPI; Man 3 -GPI, trimannosyl GPI; Man 4 -GPI, tetramannosyl GPI; PI-PLC, phospholipase C. 1160 FEBS Journal 272 (2005) 1160–1168 ª 2005 FEBS Dol-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. cerevisiae Genome 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 Candida albicans 394 37 ⁄ 70 EAK94563 Candida glabrata 433 48 ⁄ 82 CAG58266 Cryptosporidium parvum 436 23 ⁄ 71 CAD98327 Debaryomyces hansenii 428 33 ⁄ 65 CAG87189 Encephalitozoon cuniculi 393 20 ⁄ 60 NP_596980 Eremothecium gossypii 427 47 ⁄ 77 NP_984865 Gibberella zeae 423 27 ⁄ 63 EAA76896 Kluyveromyces lactis 417 47 ⁄ 81 CAG99810 Saccharomyces cerevisiae 433 – NP_009558 Schizosaccharomyces pombe 426 24 ⁄ 60 NP_592878 Magnaporthe grisea 441 23 ⁄ 59 XP_368608 Neurospora crassa 593 28 ⁄ 67 XP_325815 Ustilago maydis 485 16 ⁄ 55 EAK86320 Yarrowia lipolytica 357 31 ⁄ 70 CAG78103 Mammals Homo sapiens 493 18 ⁄ 53 NP_060307 Mus musculus 493 22 ⁄ 55 NP_848813 Rattus norvegicus 491 23 ⁄ 56 XP_345587 Plants Arabidopsis thaliana 492 22 ⁄ 52 NP_172652 Oryza sativa 486 20 ⁄ 58 AK073582 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 Giardia lamblia 381 24 ⁄ 57 EAA41032 Plasmodium falciparum 503 16 ⁄ 53 NP_701814 A L. Fabre et al. Second mannose addition to GPI precursors FEBS Journal 272 (2005) 1160–1168 ª 2005 FEBS 1161 Growth 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 YBR004c is 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 [ 3 H]inositol into proteins. The ybr004cD-pGAL- YBR004c strain was grown and labeled with [ 3 H]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 [ 3 H]inositol-labeled GPI anchored pr oteins in medium containing either galac- tose or glucose (Fig. 1C, lanes 1, 2), whereas ybr004cD- pGAL-YBR004c cells incorporated significantly less [ 3 H]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 [ 3 H]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 [ 3 H]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 Man 2 - and Man 3 -GPIs that accumulate in cells defective in A BC 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 [ 3 H]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. Second mannose addition to GPI precursors A L. Fabre et al. 1162 FEBS Journal 272 (2005) 1160–1168 ª 2005 FEBS addition 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 [ 14 C]EthN or [ 3 H]serine were therefore carried out to determine if lipid 004–1 contains an EthN-P moi- ety. To enhance [ 14 C]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 [ 14 C]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 [ 3 H]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 Man 1 (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 Man 4 -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 Man 4 -GPI isoform with JbaM would there- fore yield a GPI with a single mannose bearing EthN- P [a Man 1 (EthN-P)-GPI], whereas the minor isoform would be converted to a Man 2 (EthN-P)Man 1 -GPI. The Man 1 (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 Man 1 (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 Man 1 -GPI that may comigrate with AB CD 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 [ 3 H]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 Man 4 -GPI (lane 3) into a Man 2 (EthN-P)Man 1 -GPI and a Man 1 (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, glucosamine; PI, phosphatidylinositol. A L. Fabre et al. Second mannose addition to GPI precursors FEBS Journal 272 (2005) 1160–1168 ª 2005 FEBS 1163 [ 3 H]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 Man 3 (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 [ 3 H]inositol-labeled lipids they accumulate at 37 °C under repressing conditions were examined. At 37 °C, 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 25 °Cin 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 37 °C 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 Man 3 -GPI inter- mediate [31]. smp3–2 ⁄ ybr004cD-pGAL-YBR004c cells grown and [ 3 H]inositol-labeled in medium containing galactose prominently accumulate the Man 3 -GPI at 25 °C (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 25 °C contain predominantly lipid 004–1 and significantly less Man 3 - 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 AB 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 [ 3 H]inositol in SGalYE medium at 25 °Cor37°C (lanes 3 and 4), or in SGlcYE at 25 °Cor37°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 [ 3 H]inositol-labeled as described above after which lipids were extracted and separated by TLC. Second mannose addition to GPI precursors A L. Fabre et al. 1164 FEBS Journal 272 (2005) 1160–1168 ª 2005 FEBS family (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:: Kan R null mutation in vivo in S. cerevisiae. Heterozy- gous ybr004c::Kan R ⁄ 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:: Kan R allele 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,orSMP3, 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 [ 3 H]inositol into protein, consistent with abolition of GPI anchoring, and the accumulation of a PI-PLC- resistant, base-labile [ 3 H]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. A L. Fabre et al. Second mannose addition to GPI precursors FEBS Journal 272 (2005) 1160–1168 ª 2005 FEBS 1165 added to Man1 of GPI precursors as early as the Man 1 -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- 3 H]-myo-Inositol (sp. act. 30 CiÆmmol )1 ), [1,2– 14 C]-etha- nolamine hydrocloride and L-[ 3 H(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 synthetic 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::Kan R , YJR013- w ⁄ yjr013w::Kan R , GPI10 ⁄ gpi10::Kan R and SMP3 ⁄ smp3:: Kan R strains were purchased from Research Genetics. To construct a glucose-repressible allele of YBR004c, the YBR004c ⁄ ybr004c::Kan R heterozygous diploid was trans- formed with pGAL-YBR004c (see below). Transformants were sporulated and tetrads dissected. Haploid progeny harboring a ybr004c::Kan R 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 ⁄ ybr004cD- pGAL-YBR004c were created by mating ybr004cD-pGAL- YBR004c (MAT a, his3D1, leu2D1, ura3D0, met15D0, ybr004c::Kan R ) 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 [ 3 H]Inositol labeling of lipids in temperature-sensitive yeast strains was performed as previously described [15]. For [ 3 H]inositol or [ 3 H]serine labeling of the ybr004cD-pGAL- YBR004c strain, cells were first grown in SGlyYE medium, then shifted to SGlcYE or SGalYE medium for 16 h and labeled for 2 h at 30 °C with 15 lCi [ 3 H]inositol or 50 lCi [ 3 H]serine. [ 14 C]Ethanolamine labeling of the Dpsd1 ⁄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 [ 14 C]ethanolamine for  23 h at 25 °C. For radiolabeling of double mutant strains, cells were grown in SGlyYE medium for 2 days at 25 °C, then grown in SGalYE or SGlcYE medium for 16 h. Cells were shifted to 25 °Cor37°C for 20 min and radiolabeled with 15 lCi [ 3 H]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. [ 3 H]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. 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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. Second mannose addition to GPI precursors A L. Fabre et al. 1168 FEBS Journal 272 (2005) 1160–1168 ª 2005 FEBS . Saccharomyces cerevisiae Ybr004c and its human homologue are required for addition of the second mannose during glycosylphosphatidylinositol precursor assembly Anne-Lise. 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