Glycobiology, 1999, Vol. 9, No. 12 1287-1293
© 1999 Oxford University Press
Characterizationof an alg2 mutant of the zygomycetefungus Rhizomucor pusillus
Department of Biotechnology, Graduate School of Agricultureand Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–8657,and 2Department of Industrial Chemistry,School of Engineering, Tokai University, Kitakaname, Hiratsuka-shi,Kanagawa 259–1292, Japan
Received on January 18, 1999. revisedon May 28, 1999; accepted on May 31, 1999.
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Abstract |
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The zygomycete fungus Rhizomucor pusillus secretesan aspartic proteinase (MPP) that contains asparagine (N)-linked oligosaccharidesat two sites. Mutant strain 1116 defective in N-glycosylationsecretes MPP with truncated oligosaccharide chains. Lipid-linkedoligosaccharides in mutant 1116 were labeled with [6-3H]glucosamineand [2-3H]mannose, prepared by cyclesof solvent extraction, and analyzed by gel filtration chromatographyon a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116accumulated an intermediate, Man1GlcNAc2-dolicholpyrophosphate (PP-Dol), whereas wild-type strain F27 synthesizedthe fully assembled oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol.Consistent with this, alg2 encoding amannosyltransferase in the lipid-linked oligosaccharide biosyntheticpathway in mutant 1116 had a 5 bp insertion that generated a stop codonin the middle of the coding sequence. Transformation of mutant 1116with the intact alg2 gene on a pUC19-derived plasmidgenerated transformants that contained multicopies of alg2 atthe alg2 locus. Glycosylation of the total proteins inthe transformants was recovered to the same level as in strain F27,as determined with peroxidase-concanavalin A. These transformantsproduced MPP mainly with the same N-linked oligosaccharidesas that produced by strain F27, but still with truncated oligosaccharidesin small amounts. All of these data show that Alg2 is an
-1,3or -1,6 mannosyltransferase that elongatesMan1GlcNAc2-PP-Dol to Man2GlcNAc2-PP-Dol.The slower growth of mutant 1116 was significantly recovered onintroduction of alg2. The viability of the alg2 mutantsof the zygomycete R.pusillus makes a contrast withthe lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae. |
Introduction |
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Most secreted and membrane proteins in eukaryotes are modified bythe addition of oligosaccharides to specific asparagine residues, whichis called N-glycosylation (6
Herscovicsand Orlean, 1993; 10Kukuruzinskaand Lennon, 1994). The oligosaccharide precursor, Glc3Man9GlcNAc2,is synthesized as a lipid-linked form, which is subsequently modifiedby the action of specific endoplasmic reticulum (ER) and Golgi processingglycosidases and by Golgi glycosyltransferases. Early stages of N-linked glycosylation in the ER are remarkablyconserved through evolution of eukaryotes. alg2 mutantsof S.cerevisiae abnormally accumulate Man1GlcNAc2-PP-Doland Man2GlcNAc2-PP-Dol and exhibita temperature-sensitive lethal phenotype (Huffaker and Robbinson, 1983; 9Jackson et al., 1993).In these mutants, these two intermediates are transferred to proteinsto a significant extent, which suggests that early intermediatesmove between both faces of the ER (7Huffakerand Robbins, 1983; 8Jackson et al., 1989).
The zygomycete fungus Rhizomucor pusillus (previously called Mucor pusillus) has been used for industrial production ofa milk-clotting enzyme, Rhizomucor pepsin (MPP)(2Arima et al., 1967,1968). MPP produced by the wild-type strain contains two N-linkedglycosylation sites: Man5GlcNAc2 to Asn-79 and Man5~6GlcNAc2 toAsn-188 (16Murakami et al.,1994). We previously isolated and characterized a mutantstrain of R.pusillus defective in N-linkedglycosylation (16Murakami et al.,1994). This mutant, designated 1116, secretes a mixtureof MPP molecules, some of which contain no sugar chainand some of which contain truncated N-linked oligosaccharidechains such as Man0~1GlcNAc2. In addition,the mutant is viable, although the growth is slightly slower thanthe wild-type strain. These phenotypes led us to assume that themutation point in strain 1116 was in ALG2. We cloneda genomic DNA and cDNA encoding an ALG2 homologfrom R.pusillus (23Yamazakiet al., 1999). The cloned cDNA complementedthe temperature-sensitive growth of the alg2-1 mutantof S.cerevisiae, indicating that it representeda functional ALG2 homolog of R.pusillus. We namedthis homolog gene alg2 in accordance with the nomenclatureof fungal genes. The nucleotide sequence of alg2 cDNAfrom R.pusillus has predicted that alg2 encodesa 455-amino-acid protein showing end-to-end similarity in amino acidsequence to yeast Alg2 and containing a dolichol-binding consensussequence (Val/Ile-x-Phe-x-x-Ile, where x is any amino acid)very near its C-terminus.
In the present study, we analyzed the lipid-linked oligosaccharideaccumulated in the R.pusillus 1116 by gel filtration chromatographyand determined the mutation point by gene cloning. Consistent withthe finding that strain 1116 accumulates Man1GlcNAc2-PP-Dol, alg2 in this strain contains a 5 bp insertion inthe middle of the coding sequence, which results in generation ofa termination codon. Since strain 1116 is viable, it makes a contrastto yeast where alg2 mutations cause a lethal effect.
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Incorporation of [3H]glucosamineand [3H]mannose into the lipid-linkedoligosaccharides
We previously found that R.pusillus 1116 secreteda mixture of MPP molecules with no sugar chain and with Man0~1GlcNAc2 attwo asparagine residues (16
Murakami et al., 1994). Furthermore, analysis of glycosylationin strain 1116 by using concanavalin A and wheat germ aggulutininlectins suggested that not only MPP but also all other N-linkedglycoproteins had truncated oligosaccharide chains exposing GlcNAcresidues. For determination of lipid-linked oligosaccharides thatwere accumulated in strain 1116, we first examined incorporationof [6-3H]glucosamine and [2-3H]mannoseinto the lipid-linked oligosaccharide fraction. When [3H]glucosaminewas used, almost no accumulation of radioactivity in the "lipid" fractionwas observed in the wild-type strain F27, whereas [3H] wasrapidly incorporated and accumulated in the "oligosaccharide-lipid" fraction(Figure 1). This means that lipid-linkedoligosaccharide intermediates with short sugar chains(the lipid fraction) are rapidly converted to those with longersugar chains (the oligosaccharide-lipid fraction). On the otherhand, [3H] was rapidly incorporatedand accumulated in the lipid fraction in strain 1116, suggestingthat lipid-linked precursors were accumulated. Significant radioactivityin the oligosaccharide-lipid fraction in this mutant strain wasalso detected. Since strain 1116 was found to accumulate Man1GlcNAc2-PP-Dol(see below), this is probably due to the extraction procedure; thelipid and the oligosaccharide-lipid fractions are not strictly separated.The results from experiments with [3H]mannosewere similar (data not shown).
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Identification of lipid-linked precursor oligosaccharides
The lipid and oligosaccharide-lipid fractions labeled with [3H]glucosaminefor 2 h prepared from strains F27 and 1116 were combined and subjectedto mild acid-hydrolysis and the resultant free oligosaccharideswere resolved by chromatography on a Bio-Gel P-4 (Figure 2). The wild-type strain F27 accumulated thefully assembled precursor oligosaccharide, Glc3Man9GlcNAc2.On the other hand, the major lipid-linked oligosaccharide observedin strain 1116 was Man1GlcNAc2, as expectedfrom the above-described incorporation experiments with [3H]glucosamineand [3H]mannose.
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Determination of the mutation point in strain 1116R3
All the above data suggested that strain 1116 had a defect in the mannosyltransferasethat transfers a mannose onto Man1GlcNAc2-PP-Dolto form Man2GlcNAc2-PP-Dol. In the yeast S.cerevisiae, this enzyme is encoded by ALG2 (11
Kukuruzinska et al., 1987; 9Jackson et al., 1993).We previously cloned an ALG2 homolog from strainF27 that complemented the yeast alg2-1 mutation(23Yamazaki et al., 1999).Five introns intervene R.pusillus alg2 thatencodes 455-amino-acid protein with a dolichol-binding consensussequence at its C-terminus. On the basis of the nucleotide sequencesof the genomic alg2, we cloned the genomic alg2 regionfrom strain 1116R3. The nucleotide sequence of the genomic alg2 revealedan insertion of 5 bp, GAAGA, in the coding region, which resultsin generation of a stop codon, TAG (Figure 3).Because this sequence is repeated at this site, the mutation seemsto be generated by duplication of the 5 bp sequence. No other changesin the nucleotide sequence were found. The mutated alg2 genewould direct the synthesis of a truncated 277-amino-acid proteinthat lacks the dolichol-binding sequence.
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Complementation of the defect of strain 1116R3in N-linked glycosylation by alg2
Since the alg2 mutation of mutant 1116 was recessiveto the wild-type F27 strain, as determined by genetic studies withforced primary heterokaryons (16
Murakami et al., 1994), we introduced the genomic alg2 gene on pAP4 (Figure 4A)by transformation into an uracil auxotrophic mutant strain 1116U17derived from R.pusillus 1116R3 to see the N-linkedglycosylation of MPP and the growth in the transformants. The resultsof Southern hybridization with the alg2 cDNA(Figure 4B) and pyr4 (Figure 4C) sequences as the 32P-labeledprobe and the EcoRI-digested chromosomal DNAs fromthe transformants as the targets indicated that multiple copiesof the pAP4 sequence were inserted into the chromosome. Togetherwith the hybridization patterns obtained with the KpnI-digestedchromosomal DNA (data not shown), the pAP4 sequence was found tobe integrated at the alg2 locus by homologous recombination.Integration of these sequences into the genome is schematicallyrepresented in Figure 4D,E. Densitometricanalysis showed that the intensities of the 2.3 kb signals in transformantsRA1 and RA2 were about 6- and 5-fold, respectively, that of the7.4 kb signals (Figure 4B), indicating thatthese transformants contained 6 and 5 copies of the alg2 sequence,respectively. The analysis of the copy number on the basis of thehybridization patterns obtained with the alg2 sequencegave the same results. Transformants RA3 to RA5 also contained 3,2, and 6 copies, respectively (data not shown).
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Glycosylation of intracellular and extracellular proteins in mutant1116 occurred scarcely, as determined by concanavalin A-binding(16
Murakami et al., 1994).For binding, concanavalin A lectin requires the presence of at leasttwo -linked mannoses with free hydroxylgroups at C-3, -4, and -6. Similar analyses of glycosylated proteinsin transformant RA2 and mutant 1116R3 showed that scarce glycosylationin transformant RA2 was restored to the same level as that in thewild-type strain (Figure 5A). This findingshowed that scarce glycosylation in mutant 1116R3 was due to theframe-shift mutation in alg2.
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We then determined the patterns of N-glycosylationof MPP in these transformants grown on wheat bran medium (Figure 5B). MPP produced by strain F27 contains Man5GlcNAc2 atAsn-79 and Man5~6GlcNAc2 at Asn-188, givinga single protein band on SDS–polyacrylamide gel electrophoresis(16
Murakami et al., 1994; 17Murakami et al., 1998).MPP produced by strain 1116R3 is a mixture of three species of MPPwith different N-linked glycans; these are Man1GlcNAc2,GlcNAc2, and no sugars. These three species are visibleon SDS–polyacrylamide gel electrophoresis (16Murakami et al., 1994), although these are notvery apparent in the photograph in Figure 5B.Western blotting analysis of the MPP produced by transformants RA1 andRA2 showed that most MPP molecules are the same size as that producedby strain F27, indicating that the defect in N-glycosylationof strain 1116R3 was complemented by alg2. However,smaller amounts of MPP with Man1GlcNAc2 andno sugar were still produced. A difference in the N-glycosylation ofMPP in strain 1116R3 and the transformants is that almost no detectableamount of the MPP molecule with GlcNAc2 (correspondingto band b in Figure 5B)is produced in the latter, the reason of which is unclear at present.The glycosylation patterns of MPP produced by RA3 to RA5 were almostthe same as those produced by RA1 and RA2. The glycosylation patternwas also the same when the transformants were grown in YPD liquidmedium (data not shown). All of these data show that the defectin N-glycosylation in strain 1116R3 is complementedby alg2, but still not to the full level as inthe wild-type strain F27 in some unknown way. Strains 1116 and 1116R3 grew more slowly than strain F27 inthe range of 20°C to 48°Cboth in liquid and on solid media. The growth of transformants RA1to RA5 was significantly recovered, but still slightly slower thanin strain F27. The slow growth of the mutants strains as well asthe transformants may be caused by some additional mutation(s),since strain 1116 was derived through multiple cycles of mutagenesisfrom strain F27 for the purpose of isolating mutants producing alarger amount of MPP. It is also possible that the slow growth reflects thepartial complementation of the defect in N-glycosylation by alg2 in strain 1116R3.
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Discussion |
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The zygomycete fungus R.pusillus mutant strain1116 defective in N-glycosylation accumulates Man1GlcNAc2-PP-Dolas the lipid-linked oligosaccharide. This is consistent with theprevious observation that the largest N-glycanson MPP produced by this mutant is Man1GlcNAc2 ateither Asn-79 or Asn-188 (16
Murakami et al., 1994). In the budding yeast S.cerevisiae, alg2-1 mutants accumulated lipid-linked Man1~2GlcNAc2 at thenonpermissive temperature and the expected precursor Glc3Man9GlcNAc2 atthe permissive temperature (8Jackson et al., 1989, 1993). These observations promptedus to determine a possible mutation point(s) in the alg2 genein R.pusillus 1116R3 and to introduce an ALG2 homologof R.pusillus into the mutant. As expected, alg2 wasfound to contain a 5-bp insertion in the coding region, which resultsin translation of a truncated protein lacking the dolichol-bindingsequence. Because the defect in N-glycosylationof mutant 1116R3 was complemented by alg2 fromthe wild-type strain of R.pusillus, it is solelydue to the mutation in alg2. In mutant 1116R3, Man1GlcNAc2-PP-Dolremains without further conversion due to complete destruction of alg2. This is a contrast to the S.cerevisiaealg2-1 mutant which accumulates a small amount of Man2GlcNAc2-PP-Dolin addition to Man1GlcNAc2-PP-Dol. We assumethat the temperature-sensitive alg2-1 mutationin yeast allows addition of a mannose to Man1GlcNAc2-PP-Dol toa small extent under the conditions examined, which results in accumulationof a small amount of Man2GlcNAc2-PP-Dol. Becauseof a null mutation, but not temperature-sensitive mutation, in alg2 in mutant 1116, it accumulates Man1GlcNAc2-PP-Dolalone. Thus alg2 of R.pusillus encodes amannosyltransferase that elongates Man1GlcNAc2-PP-Dolto Man2GlcNAc2-PP-Dol. At present, it is unclearwhether Alg2 shows both the -1,3 and -1,6 mannosylation activity, as is postulatedfor Alg2 in S.cerevisiae (8Jackson et al., 1989; 6Herscovicsand Orlean, 1993).
The R.pusillus alg2 gene encoding a proteinshowing end-to-end similarity in amino acid sequence, includinga dolichol-binding sequence very near their C-termini, to yeastAlg2 complemented the temperature-sensitive growth ofthe yeast alg2-1 mutant. In addition, an aminoacid replacement at Gly-368 of the R.pusillus Alg2,generated by site-directed mutagenesis on the basis of the mutationpoints (Gly-377 to Arg) in yeast alg2-1 and alg2-2,resulted in generation of a temperature-sensitive enzyme (23Yamazaki et al., 1999).Both alg2-1 and alg2-2 contain acommon mutation at Gly-377 corresponding to Gly-368 of the fungusAlg2, in addition to one more mutation, Gln-386 to Lys for alg2-1 andGlu-54 to Lys for alg2-2. These findings implythat the R.pusillus alg2 is a functional homologof yeast ALG2. Then why does the fungus alg2 mutantaccumulate Man1GlcNAc2 exclusively, in contrastto the yeast alg2 mutants which accumulate Man2GlcNAc2 inaddition to Man1GlcNAc2? One possible explanationfor this is that the yeast mutants with point mutations in alg2 mannosylate Man1GlcNAc2-PP-Dolto a lesser extent at the nonpermissive temperature, which is thenconverted to Man2GlcNAc2-PP-Dol by a veryweak Alg2 activity. Thus, Man1~2GlcNAc2-PP-Dol aredetectable by Bio-Gel P-4 column chromatography of the lipid-linkedoligosaccharide fraction. As described above, however, it is mostlikely that the fungal Alg2 encodes an -1,3 or -1,6 mannosyltransferase that transfersmannose to Man1GlcNAc2-PP-Dol. In addition,Southern hybridization at low stringency with the alg2 sequenceas the probe excluded the possibility that R.pusillus containsan additional alg2-like sequence (data not shown).
The S.cerevisiae ALG2 gene, in addition to ALG7 and ALG1, all of which functionearly in the dolichol pathway of N-glycosylation,are essential for cell viability and perturbation in their expressioncauses G1-specific cell cycle arrest (12Lennon et al., 1995). This is a contrast withthe viability of the R.pusillus mutant 1116 havinga null mutation in alg2. Small lipid-linked glycansthat accumulate in the yeast alg1 and alg2 mutantsat the nonpermissive temperature are transferred to proteins (8Jackson et al., 1989, 1993),as in R.pusillus 1116 (16Murakami et al., 1994). Since MPP is efficientlysecreted from mutant 1116 in a large amount, the translocation ofsmaller lipid-linked oligosaccharides like Man0~1GlcNAc2-PP-Dolacross the ER membrane into the lumen and the subsequent transferof them to proteins are rather efficient. It is unclear why the R.pusillus alg2 mutant is viable but those of yeastare not. It may be related to the distant phylogeny and to totallydifferent morphogenesis (4Bartnicki-Garcia,1968; 13Lipke and Ovalle, 1998).
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Materials and methods |
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Strains and media
R.pusillus strains F27, 1116, and 1116R3 wereour laboratory stock strains (16
Murakami et al., 1994). Strain 1116 was a mutantdefective in N-linked glycosylation. Because strain 1116appeared to be an auxotrophic mutant, strain 1116R3 that grew onminimal medium and showed the same glycosylation pattern as strain1116 was derived spontaneously (16Murakami et al., 1994). pUC19 (25Yanisch-Perron et al., 1985) was used for DNA manipulationin Escherichia coli JM109 (25Yanisch-Perron et al., 1985). Fungal strains were culturedon Koji medium (10% (w/v) koji extract (obtainedfrom Meito Sangyo, Tokyo), 2% agar); PD medium(2.4% potato dextrose broth (Difco), 2% agar);and YNB medium (0.05% yeast nitrogen base without aminoacids and ammonium sulfate (Difco), 0.15% sodium glutamate,0.15% (NH4)2SO4, 1% glucose,2% agar) for uracil auxotroph strains. For the liquid culture,YPD medium (1% yeast extract (Difco), 2% bactopeptone (Difco), 2% glucose) and YNB medium were used.For uracil auxotroph strains, 400 µg/mluracil was supplemented. For MPP production, wheat bran medium (1g wheat bran (obtained from Meito Sangyo), 0.01 g (NH4)2SO4,0.8 ml distilled water per a 10 ml test tube) and YPD medium wereused. LB broth (0.5% yeast extract, 1% bacto peptone,1% NaCl) was used for E.coli.
In vivo labeling of lipid-linkedoligosaccharides
Freshly prepared spores were inoculated into glucose-restricted YPDmedium (1% yeast extract, 2% bacto peptone, 0.2% glucose) ina shaking flask. The low concentration of glucose was confirmed notto affect the growth and glycosylation. Spores had been culturedat 30°C until the germ tubes startedbranching. Portions (3 ml) were then transferred to L-shape tubescontaining 120 µCi of [3H]glucosamine(D-[6-3H]glucosaminehydrochloride, American Radiolabeled Chemicals, Inc.) or [3H]mannose(D-[2-3H]mannose, Amersham)and incubated at 30°C. Portions (150 µl) were taken out at intervals andradioactivity incorporated into all mycelia (TCA-precipitatedfraction), smaller-sized precursors (lipid fraction), and larger-sizedprecursors (oligosaccharides-lipid fraction) were measured. Proteinconcentrations were determined by the method of Lowry (1951). Protocolsfor extraction of these lipid-linked precursors were described byNishikawa (18Nishikawa, 1984, 1991).Briefly, mycelia were first extracted with CHCl3/CH3OH/H2O(1:1:1). The lower layer was saved as the lipid-fraction. The remainingmaterials were extracted with CHCl3/CH3OH/H2O(10:10:3) (the oligosaccharide-lipid fraction).
Identification of lipid-linked oligosaccharidesaccumulated in R.pusillus
The lipid fraction and the oligosaccharides-lipid fraction, obtainedafter labeling for 120 min, were combined and dried. After mildacid-hydrolysis in n-propanol and HCl, the sample wasevaporated to dryness and subjected to a Bio-Gel P-4 (-400 mesh,Bio-Rad) gel filtration column (1.0 cm diameter and 115 cm height).The standards used were Glc3Man9GlcNAc2, Man8GlcNAc2,Man5GlcNAc2, Man3GlcNAc2,Man2GlcNAc2, and GlcNAc2. Protocolsfor these procedures were described by 19Nishikawa(1991).
Determination of a mutation point in alg2 of R.pusillus 1116R3
Restriction endonucleases and T4 DNA ligase were purchased fromTakara Shuzo Co., Kyoto. General techniques for DNA manipulationin E.coli were as described by 15Maniatis et al. (1982). On the basis of the nucleotidesequence of the alg2 region in strain F27 (21Tonouchi et al., 1986; 24Yamazaki et al., 1998),two primers (5'-tttgagctcTGACTCTTGCCATTCCCGCTACTGAC-3' (the underline indicates a SacIsite; capitals represent the sequence from nucleotide positions –124to –99, when the A residue of the translational initiationcodon is taken as +1) and 5'-GCACGAAGAACAAGAATTACACGAGCGC-3' (representing the sequence from nucleotidepositions 1951 to 1978) were synthesized and used for amplificationof the whole alg2-coding sequence by the polymerasechain reaction (PCR) under the standard conditions. A 2.1 kb fragmentwas amplified as expected from the nucleotide sequence of the alg2 region. Because of the presence of a SacIsites in one of the primers and at nucleotide positions from 1755to 1760, digestion of the 2.1 kb fragment with SacIyielded a 1.9 kb fragment, which was then cloned in the SacIsite of pUC19. The cloned 1.9 kb SacI fragmentwas digested with BglII and the resulted threefragments were cloned in pUC19. The nucleotide sequences of thethree fragments were determined by the dideoxynucleotide method(20Sanger et al., 1977)using the thermo sequenase fluorescent labeled primer cycle sequencingkit (Amersham) in an Automated Fluorescence DNA sequencer (Li-Cormodel 4000L). The whole nucleotide sequence was determined withtwo independently cloned fragments to avoid errors in PCR.
Construction of pAP4 and transformation of R.pusillus 1116U17
Chromosomal DNA of R.pusillus was isolated bythe method of van Heeswijck and Roncero (1984) and purified by equilibriumcentrifugation in CsCl-ethidium bromide gradient. The genomic alg2 sequence together with its 5'-and 3'-flanking regions about 2 kb eachin length was cloned from strain F27 by the standard DNA manipulationincluding Southern hybridization and colony hybridizationwith the previously cloned alg2 cDNA sequence asthe probe (23Yamazaki et al.,1999). DNA fragments for hybridization probes were labeledwith [-32P]dCTP (110TBeq/mmol, Amersham Japan) and a BcaBESTlabeling kit (Takara Shuzo). The 2.9 kb PvuII fragmentthus cloned was introduced by transformation into strain 1116U17by the use of the host-vector system we established (23Yamazakiet al., 1999). Briefly, the PvuIIfragment was inserted into the SmaI site of pRPPyr4,resulting in pAP4 (see Figure 4). pRPPyr4is a pUC19-derived plasmid containing the R.pusillus pyr4 gene as a selection marker. pAP4 was introducedby transformation into an uracil auxotroph mutant, 1116U17, derivedby UV-mutagenesis from strain 1116R3. Among the Ura+ transformants,we chose five colonies. After two cycles of single spore isolation,we named these transformants RA1 to RA5. Integration of the pAP4sequence into the chromosome was checked by Southern hybridizationwith a 1.4 kb SacI–XbaIfragment containing the whole alg2 cDNA sequence,a 1.1 kb HincII–EcoRIfragment containing the whole pyr4 sequence, andthe full sequence of EcoRI-digested pUC19.
Immunoblotting
Spores (1 x 105) of R.pusillus strains were inoculated on wheat branmedium and incubated at 37°C for 3 days.Five milliliters of distilled water was added, and the mixture wasleft overnight at 4°C to extract MPP.The mixture was centrifuged and the supernatant was filtered througha 0.45 µm membrane filter. An appropriatevolume of the filtrate was subjected to SDS–polyacrylamidegel electrophoresis for Western blotting. The antibody specificto MPP (1Aikawa et al., 1990)was used for immunological detection of MPP by the method of 5Burnett (1981) with anti-rabbit (goat)antibodies conjugated with peroxidase (Bio-Rad) as secondary antibodies.A polyvinylidene difluoride (PVDF) membrane (Immobilon, Millipore)was used for Western blotting.
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We thank Prof. P.Robbins (Massachusetts Institute of Technology) forproviding us with yeast alg2-1 and alg2-2 mutants.This work was supported by a Grant-in-Aid for Scientific Research onPriority Areas 05274103 from the Ministry of Education, Culture,and Science of Japan.
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Abbreviations |
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MPP, Rhizomucor pusillus pepsin;PP-Dol, dolichol pyrophosphate; ER, endoplasmic reticulum.
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a To whom correspondenceshould be addressed
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References |
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