Sequence analysis of SLA2 of the dimorphic yeasts Candida albicans and Yarrowia lipolytica.

We report the complete nucleotide sequence of SLA2 of the dimorphic yeasts Candida albicans and Yarrowia lipolytica. In Saccharomyces cerevisiae, SLA2 codes for an actin binding protein. The deduced amino acid (aa) sequences of C. albicans CaSla2p and Y. lipolytica YlSla2p consist of 1063 and 1054 aa, respectively. The alignment of the deduced proteins of Saccharomyces cerevisiae, Y. lipolytica and C. albicans shows regions of identity in the N-terminal part of the proteins, which are essential for growth at 37 degrees C, endocytosis and actin organization in S. cerevisiae. The Sla2p proteins have also several conserved regions in the C-terminal moiety, the I/LWEQ boxes, displaying homology to the talin protein of mouse, Dictyostelium discoideum, Caenorhabditis elegans and to human huntingtin interacting protein (Hip 1p). The sequence data of C. albicans SLA2 are registered in the EMBL database (AJ009556), and for the Y. lipolytica gene in GenBank (U65409).     

Molecular cloning and characterization of a glucan synthase gene from the human pathogenic fungus Paracoccidioides brasiliensis

1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.     

Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis

Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.     

A single FKS homologue in Yarrowia lipolytica is essential for viability

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall which gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role. FKS1 has been characterized in Saccharomyces cerevisiae, where its function is at least partially redundant with that of FKS2/GSC2. FKS homologues have also been identified in several other fungal species, including Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Cryptococcus neoformans and Paracoccidiodes brasiliensis. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Fks1ps to isolate the possible FKS homologue genes of the strictly aerobic non-conventional yeast Yarrowia lipolytica. Using this approach, we have isolated a single FKS homologue which we have named YlFKS1; this codes a 1961 amino acid protein that shows a high degree of homology with other Fksps. Expression analysis of YlFKS1 under different conditions affecting the cell wall did not reveal significant differences. Finally, attempts to obtain a Y. lipolytica strain containing a disrupted YlFKS1 allele failed, despite having used two different techniques. Taken together, these results suggest that, unlike S. cerevisiae, YlFKS1 is the only FKS1 homologue in Y. lipolytica and is essential for growth.     

Isolation of the MNN9 gene of Yarrowia lipolytica (YlMNN9) and phenotype analysis of a mutant ylmnn9 Delta strain

In this work we describe the isolation of the Yarrowia lipolytica homologue of Saccharomyces cerevisiae MNN9 gene, which we have named YlMNN9, and the phenotype analysis of a Y. lipolytica strain containing the disrupted YlMNN9 allele. YlMNN9 was cloned using degenerate consensus oligonucleotides to generate specific probes that were in turn used to screen mini-gene libraries. The gene is defined by a 1014 bp ORF predicted to encode a protein 337 amino acids long that shares significant homology with the Mnn9ps of S. cerevisiae, Candida albicans and Hansenula polymorpha, including a putative N-terminal transmembrane domain. Disruption of YlMNN9 leads to phenotypes such as resistance to sodium orthovanadate and sensitivity to hygromycin B, compatible with a glycosylation defect, and hypersensitivity to Calcofluor white, Congo red or zymolyase, characteristic of cell wall defects. Analysis of cell wall proteins present in beta-mercaptoethanol and zymolyase extracts showed significant differences between the parental and the ylmnn9 Delta strain. These results suggest that, as has been the case with the mnn9 strain of S. cerevisiae, the ylmnn9 Delta strain we present in this work, could be used to study the cell wall proteins of Y. lipolytica and how they are organized into the cell wall.     

Antibodies directed against a yeast carboxyl-terminal peroxisomal targeting signal specifically recognize peroxisomal proteins from various yeasts.

The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme (hydratase-dehydrogenase-epimerase) to peroxisomes of both Candida albicans and Saccharomyces cerevisiae (Aitchison,J.D., Murray, W.W. and Rachubinski, R. A. (1991).J. Biol. Chem. 266, 23197-23203). We investigated the possibility that this tripeptide may act as a general peroxisomal targeting signal (PTS) for other proteins in the yeasts C. tropicalis, C. albicans, Yarrowia lipolytica and S. cerevisiae, and in rat liver. Anti-AKI antibodies raised against the carboxyl-terminal 12 amino acids of trifunctional enzyme were used to search for this PTS in proteins of these yeasts and of rat liver. The anti-AKI antibodies reacted exclusively with multiple peroxisomal proteins from the yeasts C. tropicalis, C. albicans and Y. lipolytica. There was a weak reaction of the antibodies with one peroxisomal protein from S. cerevisiae and no reaction with peroxisomal proteins from rat liver. Antibodies directed against a synthetic peptide containing a carboxyl-terminal Ser-Lys-Leu PTS (Gould, S. J., Krisans, S., Keller, G.-A. and Subramani, S. (1990). J. Cell Biol. 110,27-34) reacted with multiple peroxisomal proteins of rat liver and with peroxisomal proteins of yeast distinct from those identified with anti-AKI antibodies. These results provide evidence that several peroxisomal proteins of different yeasts contain a PTS antigenically similar to that of C. tropicalis trifunctional enzyme and that this signal is absent from peroxisomal proteins from at least one mammalian system, rat liver.     

Cloning and sequence of the LYS2 homologue gene from the osmotolerant yeast Pichia sorbitophila

We have isolated the Pichia sorbitophila LYS2 (PsLYS2) gene by complementation of a lys2 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 4197 bp and the deduced translation product shares a global identity of 66% and 58% to the Lys2 protein homologues of Candida albicans and S. cerevisiae, respectively. Analysis of PsLYS2 sequence suggests that, similarly to S. cerevisiae LYS2, it codes for a polypeptide having two separate enzymatic activities which reside in different domains of the protein, including an adenylate domain, an acyl-carrier site and a short-chain reductase domain. Several GCN4- and NIT2-binding motifs have been matched in the promotor sequence of PsLYS2. In addition, upstream of the sequenced PsLYS2 sequence, we have found the 3'-terminal half of a gene of same orientation encoding a RAD16-like protein, a genomic organization similar to that of C. albicans.     

Gene order in a 10 275 bp fragment of Yarrowia lipolytica, including adjacent YlURA5 and YlSEC65 genes conserved in four yeast species.

We have determined the sequence of a 10275 bp DNA segment of Yarrowia lipolytica located on chromosome VI. The sequence contains six complete open reading frames (ORFs) longer than 100 amino acids and two more partial ORFs at both ends. Two of the ORFs encode for the well-characterized genes YlURA5 (orotate phosphoribosyltransferase) and YlSEC65 (encoding a subunit of the signal recognition particle). These two genes show an identical organization-located on opposite strands and in opposite orientations-in four yeast species: Saccharomyces cerevisiae, Kluyveromyces lactis, Candida albicans and Y. lipolytica. One ORF and the two partial ORFs code for putative proteins showing significant homology with proteins from other organisms. YlVI-108w (partial) and YlVI-103w show 39% and 54% identity, respectively, with YDR430c and YHR088w from S. cerevisiae. YlVI-102c (partial) shows significant homology with a matrix protein, lustrin A from Haliotis rufescens, and with the PGRS subfamily (Gly-rich proteins) of Mycobacterium tuberculosis. The three remaining ORFs show weak or non-significant homology with previously sequenced genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AI006754.     

Septin localization in the dimorphic fungus Paracoccidioides brasiliensis

Septins are evolutionarily conserved proteins that contain a GTPase domain and are capable of forming filaments at the cell periphery. Septins are involved in many essential cellular processes, such as cytokinesis and cell polarization, and are used as markers of morphogenesis in several fungi. Dimorphism in fungi enables cells to switch between morphologies (yeast or filament forms), due to changes in the temperature of the environment. We analysed the localization of septin proteins in yeast and filamentous cells of the dimorphic fungus Paracoccidioides brasiliensis, a common cause of granulomatous mycosis. In order to determine septin localization, we first cloned Cdc12p, a septin homolog from P. brasiliensis, and expressed it in Escherichia coli. Following PbCdc12p purification, specific serum against PbCdc12p were raised for use in immunofluorescence assays. We observed the hourglass and ring forms of septin filaments during cell division in yeast. Septin filaments were also simultaneously localized in the necks of multiple budding cells. A distinctive pattern of punctuate and/or diffuse localization was also seen in the periphery of multinucleate yeast cells and at the tips and septa of filamentous cells. A more diffuse and punctuate pattern of localization observed in P. brasiliensis cells seems to be unique to filamentous and dimorphic fungi and may be related to their specialization in cell wall deposition, morphogenesis and cell cycle control.     

The ALS5 gene of Candida albicans and analysis of the Als5p N-terminal domain

ALS genes of Candida albicans encode a family of cell-surface glycoproteins with a three-domain structure. Each Als protein has a relatively conserved N-terminal domain, a central domain consisting of a tandemly repeated motif, and a serine-threonine-rich C-terminal domain that is relatively variable across the family. The ALS family exhibits several types of variability that indicate the importance of considering strain and allelic differences when studying ALS genes and their encoded proteins. Analysis of ALS5 provided additional evidence of variability within the ALS family. Comparison of the ALS5 sequence from two strains indicated sequence differences larger than strain or allelic mismatches observed for other C. albicans genes. Screening a collection of commonly used C. albicans strains and clinical isolates indicated that ALS5 is not present in several of these strains, supporting the conclusion that the Als protein profile is variable among C. albicans isolates. Physical mapping of ALS5 showed that it is located close to ALS1 on chromosome 6. The N-terminal domain of Als5p was produced in Pichia pastoris to initiate structural analysis of this portion of the protein. The hydrophobic character of this portion of the protein was exploited in the purification scheme. Circular dichroism analysis of the purified, authenticated protein yielded a high content of antiparallel beta-sheet and little to no alpha-helical structure. These results are consistent with the conclusion that the N-terminal domain of Als5p has an immunoglobulin fold structure similar to that found in many cell adhesion molecules. Gene sequences of C. albicans ALS5 (Accession No. AF068866) and TPI1 (Accession No. AF124845) have been deposited in the GenBank database.     

蛋白组学差异揭示黑曲霉（Aspergillus niger）高产柠檬酸的分子机制

黑曲霉（Aspergillus niger）YX-1217是一株典型的柠檬酸高产菌株,基于二维聚丙烯酰胺凝胶电泳（2-DE）对黑曲霉YX-1217 和退化菌株YX-1217G的胞外分泌蛋白进行比较,同时利用同位素标记相对和绝对定量（iTRAQ）技术比较了两株菌的胞内蛋白差 异,揭示黑曲霉YX-1217高产柠檬酸的分子机制。 结果显示,菌株黑曲霉YX-1217G的胞外蛋白数量和浓度均显著低于菌株黑曲霉 YX-1217（P＜0.05）,通过质谱鉴定了6个黑曲霉YX-1217高表达蛋白;利用iTRAQ技术共鉴定到3 553个蛋白,将差异蛋白按照京都基 因与基因组百科全书（KEGG）代谢数据库进行分类,发现与柠檬酸生物合成相关的酶在菌株黑曲霉YX-1217中的表达量显著高于退 化菌株（P＜0.05）,这些蛋白的高效表达是菌株黑曲霉YX-1217高产柠檬酸的原因之一,为开发柠檬酸或其他有机酸的高产菌株提供 新的思路和观点。     

RNA干扰技术降低黑曲霉细胞工厂的内源蛋白背景

利用RNA干扰技术(RNA interference,RNAi)改造黑曲霉细胞工厂,减少其内源蛋白表达背景。研究RNAi载体中反向互补片段特异性对RNA干扰的影响,同步干扰糖化酶基因glaA、淀粉酶基因amyA及蛋白酶调控因子基因prtT,并利用经RNA干扰改造的黑曲霉宿主SH-2表达异源脂肪酶基因tll。研究结果表明,不同长度的靶基因反向互补片段表现出不同程度的RNA干扰效应,载体pAMDS-RNAi-glaA388对糖化酶的干扰效果(94.40%)比载体pAMDS-RNAi-glaA784(70.60%)好;经三基因RNAi载体pAMDS-multi-RNAi改造的黑曲霉菌株中glaA、amyA、prtT的表达量仅为原始菌株的5.30%、17.10%和34.60%;利用经三基因RNAi载体改造的黑曲霉菌株表达脂肪酶tll,最高酶活达到97.27 U/mL,比利用原始菌株表达tll的最高酶活(73.05 U/mL)提高33.16%。因此,RNAi技术可以有效降低黑曲霉宿主内源蛋白的表达背景,有利于提高外源基因在黑曲霉细胞工厂中的表达水平和稳定性。     

Characterization of a disulphide-bound Pir-cell wall protein (Pir-CWP) of Yarrowia lipolytica

In this work we have studied the disulphide-bound group of cell wall mannoproteins of Yarrowia lipolytica and Candida albicans. In the case of Y. lipolytica, SDS-PAGE analysis of the beta-mercaptoethanol-extracted material from the purified cell walls of the yeast form, showed the presence of a main polypeptide of 45 kDa and some minor bands in the 100-200 kDa range. This pattern of bands is similar to that obtained in identical extracts in Saccharomyces cerevisiae (Moukadiri et al., 1999), and besides, all these bands cross-react with an antibody raised against beta-mercaptoethanol-extracted material from the purified cell walls of S. cerevisiae, suggesting that the 45 kDa band could be the homologue of Pir4 of S. cerevisiae in Y. lipolytica. To confirm this possibility, the amino-terminal sequences of two internal regions of the 45 kDa protein were determined, and degenerate oligonucleotides were used to clone the gene. The gene isolated in this way codes a 286 amino acid polypeptide that shows homology with the Pir family of proteins of S. cerevisiae (Russo et al., 1992; Toh-e et al., 1993), accordingly we have named this gene YlPIR1. Disruption of YlPIR1 led to a slight increase in the resistance of the cells to calcofluor white, Congo red and zymolyase, but did not cause changes in cell morphology, growth rate or morphological transition.     

Cloning of a fatty acid synthase component FAS1 gene from Saccharomyces kluyveri and its functional complementation of S. cerevisiae fas1 mutant

A gene encoding a fatty acid synthase component, FAS1, has been cloned from a genomic library of the polyunsaturated fatty acid (PUFA)-producing yeast Saccharomyces kluyveri. This gene (named Sk-FAS1) was found to contain an open reading frame of 6150 bp, coding for 2049 amino acids. The deduced Sk-FAS1 protein showed significant (75-59%) homology with FAS proteins from the other yeasts, including S. cerevisiae, Candida albicans and Yarrowia lipolytica. The substrate-binding sites of the acetyl transferase and malonyl/palmitoyl transferase domains, and the FMN- and NADPH-binding sites of the enoyl reductase domain, were all highly conserved. Expression of the Sk-FAS1 gene in S. cerevisiae complemented genetic disruption of the S. cerevisiae FAS1 gene (Sc-FAS1), suggesting the formation of a heterogeneous complex of Sk-FAS1 (beta) and Sc-FAS2 (alpha), which is able to function to synthesize fatty acids. Compared with the isogenic wild-type of S. cerevisiae, as well as S. kluyveri, the S. cerevisiae fas1 mutant carrying the Sk-FAS1 gene showed an increase in the relative amount of 16-carbon fatty acids and a decrease in 18-carbon fatty acids.     

Genome-wide identification of fungal GPI proteins

Glycosylphosphatidylinositol-modified (GPI) proteins share structural features that allow their identification using a genomic approach. From the known S. cerevisiae and C. albicans GPI proteins, the following consensus sequence for the GPI attachment site and its downstream region was derived: [NSGDAC]-[GASVIETKDLF]-[GASV]-X(4,19)-[FILMVAGPSTCYWN](10)>, where > indicates the C-terminal end of the protein. This consensus sequence, which recognized known GPI proteins from various fungi, was used to screen the genomes of the yeasts S. cerevisiae, C. albicans, Sz. pombe and the filamentous fungus N. crassa for putative GPI proteins. The subsets of proteins so obtained were further screened for the presence of an N-terminal signal sequence for the secretion and absence of internal transmembrane domains. In this way, we identified 66 putative GPI proteins in S. cerevisiae. Some of these are known GPI proteins that were not identified by earlier genomic analyses, indicating that this selection procedure renders a more complete image of the S. cerevisiae GPI proteome. Using the same approach, 104 putative GPI proteins were identified in the human pathogen C. albicans. Among these were the proteins Gas/Phr, Ecm33, Crh and Plb, all members of GPI protein families that are also present in S. cerevisiae. In addition, several proteins and protein families with no significant homology to S. cerevisiae proteins were identified, including the cell wall-associated Als, Csa1/Rbt5, Hwp1/Rbt1 and Hyr1 protein families. In Sz. pombe, which has a low level of (galacto)mannan in the cell wall compared to C. albicans and S. cerevisiae, only 33 GPI candidates were identified and in N. crassa 97. BLAST searches revealed that about half of the putative GPI proteins that were identified in Sz. pombe and N. crassa are homologous to known or putative GPI proteins from other fungi. We conclude that our algorithm is selective and can also be used for GPI protein identification in other fungi.     

12种真菌钾离子通道蛋白生物信息学分析

利用生物信息学对真菌钾离子通道蛋白进行序列分析,推断并预测其结构和功能。本文筛选了酿酒酵母、黄曲霉、烟曲霉、禾谷镰刀菌、玉米黑粉菌、布拉克须霉等12种真菌的钾离子通道蛋白,综合采用Blast、ProtParam、SignalP和Swiss-Model等生物信息学网站和软件对这些蛋白的理化性质、系统进化、蛋白结构和功能域等进行了分析。结果表明:所选择的钾离子通道蛋白氨基酸序列长度在659~928 aa之间,理论等电点在5.45~9.54之间,主要定位在质膜、液泡上。二级结构分析表明,真菌钾离子通道蛋白均由α-螺旋、β-折叠、无规则卷曲和延伸链等结构元件组成,其空间结构高度相似,且12种钾离子通道蛋白都拥有保守的TxGYGD钾离子选择器特征片段。     

Direct contact membrane inoculation of yeasts and moulds for evaluating preservative efficacy in solid cosmetics

A direct contact membrane inoculation technique for yeasts and moulds was used to evaluate the preservation efficacy and antimicrobial activity of Germall 115 and Germall II in pressed eye shadows. Test organisms on membrane filters were placed in direct contact with cosmetics at room temperature under humid conditions. Growth on membranes was removed daily, or as appropriate, and cultured on potato dextrose agar containing lecithin and Tween 80. Linear regression analysis was used to determine product preservation efficacy. Average D values of 1 and 3 days for Candida albicans American Type Culture Collection (ATCC) 10231 and 17 and 29 days for Aspergillus niger ATCC 16404 were obtained on two eye shadows we prepared (in-house eye shadows) with parabens and either Germall II or Germall 115 as preservatives. A decimal reduction time (D value) of 6–7 days was calculated for the yeast on a commercial eye shadow preserved with parabens and Germall 115. A. niger multiplied on six of seven replicates of this commercial product to attain a nearly 3 log₁₀ increase in 20 days. On one replicate, A. niger showed a 1 log₁₀ increase in the first 10 days, and then decreased linearly (r =— 0.95) to <10 colony-forming units per membrane by day 24. The method used with C. albicans and A. niger was then used with bacteria. The method was sensitive enough to differentiate the antimicrobial activity of the Germall 115 and Germall II against fungi but not against bacteria. The in-house and commercial products were preserved most effectively against the three bacteria tested and least effectively against the mould.     

Cloning and characterization of WMSU1, a Williopsis saturnus var. mrakii gene encoding a new yeast SUN protein involved in the cell wall structure

SUN proteins of Saccharomyces cerevisiae have been defined on the basis of high homologies in their C-terminal domain. Recently, two of these four proteins were shown to be involved in cell wall morphogenesis (Mouassite et al., 2000a). In the present study, we have isolated WMSU1 (Accession No. AF418983), a new SUN-related gene, from W. saturnus var. mrakii MUCL 41968. Sequencing of the gene revealed an open reading frame coding for 402 amino acids. The predicted amino acid sequence of WMSU1 is closely related to the S. cerevisiae SUN proteins and to other yeast proteins involved in cell wall metabolism. WMSU1 is proposed to encode a cell wall protein since its predicted product contains a signal sequence, a Kex2p cleavage site and a serine/threonine-rich N-terminal domain. Southern blot analysis of the W. saturnus var. mrakii MUCL 41968 genome using the highly conserved domain of WMSU1 as a probe suggested that the isolated gene belongs to a multigenic family. Expression of WMSU1 in E. coli led to a 45 kDa protein, which appeared to be toxic to this host. Scanning electron microscopy analysis of a recombinant S. cerevisiae producing Wmsu1p showed that this strain exhibited an altered cell wall, thus pointing to a probable role of this protein in the cell wall structure.     

High expression of a synthetic gene encoding potato alpha-glucan phosphorylase in Aspergillus niger

We describe the successful heterologous expression of the Solanum tuberosum alpha-glucan phosphorylase (GP) gene in Aspergillus niger. Special attention was paid to the influence of different codon usage and A+T content in the coding region on GP protein expression. Use of A. niger-preferred codon usage and lower A+T content in a synthetic gene (GP-syn) resulted in a significant improvement in the level of the GP mRNA and a dramatic increase in the quantity of GP protein produced such that it accounted for approximately 10% of the total soluble protein. We suggest that redesigning the primary DNA sequence encoding a desired protein product can be an extremely effective method for improving heterologous protein production in filamentous fungi.     

Yarrowia lipolytica SRP receptor alpha-subunit

The Yarrowia lipolytica SRP101 homologue encoding the signal recognition particle (SRP) receptor alpha-subunit (SRalphap) was cloned using degenerate primers designed for conserved GTP-binding domains. Sequencing of 2814 nucleotides revealed an open reading frame of 1671 base pairs encoding a putative protein of 557 amino acids with a predicted molecular mass of 61 kDa. Like other SRP101 homologues, Y. lipolytica SRP101 contains a highly conserved C-terminal GTP binding site. It has 44%, 34% and 22% sequence identity with S. cerevisiae, mammalian and Escherichia coli homologues, respectively. As found for SRP protein subunits of Y. lipolytica, SRP101 is important but not essential for cell growth. A conditional mutation in SRP101 affected synthesis/translocation of alkaline extracellular protease and Kar2p consistent with Srp101p functioning as an SRP receptor subunit. The SRP101 sequence has been deposited in GenBank under Accession No. AF132597.