A comparative immunological investigation of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa

The immunological relations of the cytochrome P-450 from the n-alkane utilizing yeast Candida maltosa to cytochrome P-450 forms of other organisms - yeasts, bacteria and mammalia - were investigated using a solid-phase double-antibody radioimmunoassay. Only the microsomal fraction of other n-alkane utilizing yeasts shows a distinct cross-reaction with an antiserum against cytochrome P-450 from Candida maltosa. Neither the tested bacterial nor the mammalian cytochromes P-450 cross-react with the antiserum.     

Physiological and DNA characterization of Candida maltosa, a hydrocarbon-utilizing yeast.

Selected yeast classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake, Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.     

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.     

New yeast vectors containing the autonomously replicating sequences from Candida maltosa genome

Two different DNA sequences from the yeast Candida maltosa confer the ability to replicate autonomously to the yeast integrative vector pLD700 on which they are cloned. The recombinant plasmids pLD701 and pLD702 with autonomously replicating sequences (ARS) from Candida maltosa and LEU2 gene from Saccharomyces cerevisiae transform the auxotrophic strain S. cerevisiae DC5 with the efficiency 3-5 x 10(3) per microgram of DNA. Like other yeast vectors harbouring ARS, these plasmids are not stable in yeast cells. Restriction and hybridization analyses have revealed the pLD701 plasmid to contain ARS from chromosomal DNA of C. maltosa. Plasmid pLD701 appears to be a useful vector for yeast transformation.     

Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum

cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2. The amino acid sequences deduced were about 55% identical. Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences. A massive proliferation of the endoplasmic reticulum was observed in the S. cerevisiae cells which produced P-450. Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm. As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.     

Phylogenetic identification of n-alkane assimilating Candida yeasts based on nucleotide divergence in the 59 end of LSU rDNA gene

Phylogenetic relationships of several species within the n-alkane assimilating Candida yeasts were investigated by using characters from the nucleotide sequence of the variable D1/D2 region at the 5' end of a large-subunit (26S) ribosomal DNA (rDNA) gene. First the nucleotide sequences of D1/D2 domain of Candida sp. 1098 (formerly identified as C. tropicalis 1098) and its dicarboxylic acid-producing-mutant strain M1210 were investigated. These two nucleotide sequences were identical and lacked only one base pair compared with that of C. maltosa CBS 5611 (type strain), and they were identified as C. maltosa. We then showed that C. maltosa IFO 1978 (formerly identified as C. cloacae) and C. maltosa IFO 1975 (formerly identified as C. subtropicalis) had the same nucleotide sequence and had only one base pair substitution compared with C. maltosa CBS 5611 (type strain), which is consistent with conventional classification. We also found that another widely studied n-alkane assimilating Candida yeast, C. tropicalis pk233, to be C. viswanathii.     

表型相似的热带假丝酵母和麦芽糖假丝酵母在脉冲电泳核型上的差异

热带假丝酵母Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ （Ｃａｓｔｅｌｌａｎｉ ）Ｂｅｒｋｈｏｕｔ和麦芽糖假丝酵母Ｃ． ｍａｌｔｏｓａＫｏｍａｇａｔａ, Ｎａｋａｓｅ ＆ Ｋａｔｓｕｙａ是两种可利用烃类作为碳和能量来源的酵母菌,前者还是一种条件致病菌,可引起系统感染。这两种假丝酵母菌在形态和生理生化性状上非常相似,用常规分类方法不易准确地鉴别。本研究对Ｃ． Ｔｒｏｐｉｃａｌｉｓ和Ｃ ｍａｌｔｏｓａ的模式菌株以及中国普通微生物菌种保藏中心（ＣＧＭＣＣ）保藏的归于这两个种名下的其它菌株进行了脉冲电泳核型比较分析。发现这两个表型相似的种具有明显不同的染色体ＤＮＡ分子带型,而同一种内的不同菌株却具有相同或相似的分子核型。Ｃ．Ｔｒｏｐｉｃａｌｉｓ的特异染色体ＤＮＡ分子带谱为２条８．５—１．２ Ｍｂ的带, ４条２．３－３．４ Ｍｂ的带。 Ｃ ｍａｌｔｏｓａ的特异带谱为： ３～４条分子量在１．１－１．３Ｍｂ范围内的带, １条约为２．２Ｍｂ的带以及２－３条大小为３．２－３．５Ｍｂ的带。 Ｃ ｔｒｏｐｉｃａｌｉｓ与Ｃ ｍａｌｔｏｓａ在染色体ＤＮＡ分子带型上的差异与二者在可溶性淀粉的同化能力和４０℃下的生长能力上的差异具有明显的相关性…     

Ray38p, a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, is a ribosome-associated protein and dissociated from ribosomes prior to the induction of cycloheximide resistance in Candida maltosa

Cycloheximide (CYH) resistance in Candida maltosa is dependent on the induction of a ribosomal protein, Q-type L41, the 56th residue of which is glutamine, not proline as in ordinary P-type L41. We found that a 38-kDa protein in a wild-type C. maltosa ribosomal fraction became undetectable upon CYH treatment but detectable again with the establishment of CYH resistance by the induction of Q-type L41. We cloned a gene coding for this protein and named it RAY38 (ribosome-associated protein of yeast). Ray38p is a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, and has a putative RNA-binding motif RGG. The ribosome-associated Ray38p was phosphorylated at serine and threonine residues, and Ray38p that was dissociated from ribosome by CYH treatment was highly phosphorylated in threonine residues. A ray38 null mutant recovered faster from CYH-caused growth stasis than the wild-type strain, suggesting that the dissociation of Ray38p from ribosome facilitates the induction of CYH resistance in C. maltosa.     

In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor

The in vitro synthesized precursor of the alpha-factor pheromone, prepro-alpha-factor, of Saccharomyces cerevisiae was translocated across yeast microsomal membranes in either a homologous or a wheat germ cell free system. Translocated prepro-alpha-factor was glycosylated, sedimented with yeast microsomal vesicles, and was protected from digestion by added protease, but was soluble after alkaline sodium carbonate treatment. Thus prepro-alpha-factor was properly sequestered within yeast microsomal vesicles, but was not integrated into the lipid bilayer. In marked contrast to protein translocation across mammalian microsomal membranes, translocation of prepro-alpha-factor across yeast microsomal membranes could occur posttranslationally. This reaction required protein components in the yeast microsomal fraction that could be inactivated by alkylation or proteolysis, was ATP-dependent, and was insensitive to the presence of a variety of uncouplers and ionophores.     

Sequences of two tandem genes regulated by carbon sources, one being essential for n-alkane assimilation in Candida maltosa

Several n-alkane-inducible clones were isolated from the genomic library of an n-alkane-assimilation yeast, Candida maltosa, by the differential hybridization method. Among these, one of the most predominantly expressed clones was analyzed. The nucleotide sequence of the cloned DNA fragment showed that it contained two open reading frames, one encoding a protein of 127 amino acids (aa) and the other a protein of 276 aa. The former was named POX18Cm, because the sequence was highly homologous to that of the Candida tropicalis gene, POX18, which already had been identified as encoding a small oleate-inducible peroxisomal protein. The latter, named ALI1, had no homologous sequences in the EMBL database (1990 release). Northern-blot hybridization indicated that the expression of these two genes was regulated by carbon sources in the media. From gene-disruption experiments, it was concluded that ALI1 was essential for assimilation of n-alkane by C. maltosa.     

Inducible expression of a gene encoding an L41 ribosomal protein responsible for the cycloheximide-resistant phenotype in the yeast Candida maltosa.

In a previous paper (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262, 1992), we showed that in each genome of several yeast species, there is one of two types of L41 gene, one for an L41 (Q-type) protein which confers cycloheximide (CYH) resistance or one for an L41 (P-type) protein which does not. These genes have been suggested to be responsible for the CYH response used in taxonomy. For example, Saccharomyces cerevisiae, which is CYH sensitive, has a P-type L41 gene, while Kluyveromyces fragilis and Candida maltosa, which are CYH resistant, have Q-type L41 genes. However, in contrast to K. fragilis, which is constitutively resistant to CYH, C. maltosa is inducibly resistant to CYH. Here, we show that C. maltosa has both types of the L41 gene in its genome and that expression of the Q-type L41 gene is induced by CYH while the P-type L41 gene is constitutively expressed.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

Incorporation of galactose from UDP-galactose into microsomal and golgi membranes of rat liver

Rough and smooth microsomes and Golgi membranes were incubated with UDP[¹⁴C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from β-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.     

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.     

Isolation and characterization of α-aminoadipate-δ-semialdehyde overproducing mutants from yeasts

Abstract We isolated from Candida maltosa mutants lacking saccharopine reductase ( lys9 ) and saccharopine dehydrogenase ( lys1 ). They accumulated α-aminoadipate-δ-semialdehyde (AASA) in the cell and excreted it into the culture medium. In the presence of 15 g glucose/l, 1.25 g NH₄H₂PO₄/l and 50 mg l -lysine/l in a minimal salt medium C. maltosa G285 ( lys1 ) produced about 80–90 mg AASA/l within 48 h. It is the first report of lysine-requiring yeast mutants that accumulate and excrete AASA. In contrast, Pichia guilliermondii lys9 mutants lacked this AASA overproduction. The AASA accumulation by C. maltosa mutants may be explained by the low feedback regulation of their homocitrate synthase and the equilibrium of the enzyme reactions involved in the lysine biosynthesis.     

Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.     

The nascent polypeptide-associated complex (NAC) of yeast functions in the targeting process of ribosomes to the ER membrane.

We study here the binding of ribosomes to the endoplasmic reticulum (ER) membrane and its dependence on nascent polypeptide-associated complex (NAC). For this, we use an in vitro translation system in combination with isolated microsomes. Importantly, all components in the system are derived from a single source, Saccharomyces cerevisiae. Ribosome nascent chains (RNCs) of the two naturally occurring invertase species (secreted or cytosolic) were prepared in wild-type, delta alpha NAC or delta alpha beta 1 beta 3 NAC translation lysates and tested for binding to the corresponding microsomal membranes. We provide evidence that NAC prevents binding of RNCs without a signal sequence to yeast membranes. In the absence of NAC, signal-less RNCs are able to bind to ER membranes. However, following puromycin treatment, only very few nascent chains translocate into the lumen, as detected by glycosylation.     

Biogenesis of microsomal membrane glycoproteins in rat liver. II. Purification of soluble glycoproteins and their incorporation into microsomal membranes

Sialoproteins isolated from the soluble fraction of rat liver could be incorporated into microsomal membranes. This incorporation was dependent on protein concentration, time, and temperature. Sodium dodecyl sulfate gel electrophoresis of membrane proteins after in vitro incorporation showed four major sugar-containing peaks and was similar to that found after in vivo labeling. Most of the incorporated protein was tightly bound to the microsomal membrane. Gel filtration and ion-exchange chromatography revealed the presence of several cytosolic glycoproteins that could be incorporated into microsomes. During prolonged centrifugation in a KBr solution with a density of 1.21 a highly labeled ([3H]glucosamine) protein (mole wt approximately to 70,000) that was actively incorporated into microsomes could be recovered in the upper region of the tube. These results demonstrate that several cytoplasmic glycoproteins of rat liver are transferred into microsomal membranes and that one of these is a lipoprotein.     

Hydroxylation of phenol to catechol by Candida tropicalis: involvement of cytochrome P450

Microsomal preparations isolated from yeast Candida tropicalis (C. tropicalis) grown on three different media with or without phenol were isolated and characterized for the content of cytochrome P450 (CYP) (EC 1.14.15.1). While no CYP was detected in microsomes of C. tropicalis grown on glucose as the carbon source, evidence was obtained for the presence of the enzyme in the microsomes of C. tropicalis grown on media containing phenol. Furthermore, the activity of NADPH: CYP reductase, another enzyme of the microsomal CYP-dependent system, was markedly higher in cells grown on phenol. Microsomes of these cells oxidized phenol. The major metabolite formed from phenol by microsomes of C. tropicalis was characterized by UV/vis absorbance and mass spectroscopy as well as by the chromatographic properties on HPLC. The characteristics are identical to those of catechol. The formation of catechol was inhibited by CO, the inhibitor of CYP, and correlated with the content of cytochrome P450 in microsomes. These results, the first report showing the ring hydroxylation of phenol to catechol with the microsomal enzyme system of C. tropicalis, strongly suggest that CYP-catalyzed reactions are responsible for this hydroxylation. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by the C. tropicalis strain.     

Microsomal chitinase activity from Candida albicans

Chitinase (E.C. 3.2.1.14) was characterized in microsomal fractions from yeast cells of Candida albicans. Following six washes with buffer (50 mM Bis-Tris.Cl, pH 6.5), enzyme activity of microsomes fell markedly to 0.3% of total and 6% of the specific activity detected in the low-speed supernatant (9000 X g) of a cell lysate. An apparently zymogenic, microsomal chitinase activity became more readily detectable with washing and after six washes enzyme activity was activated 1.7-fold following pre-incubation with trypsin. The following properties of microsomal chitinase were closely comparable with those for cytosolic chitinase (indicated in parentheses): Km = 2.1 mg chitin per ml (2.9 mg chitin per ml); temperature optimum = 45 degrees C (45 degrees C); inhibition by allosamidin competitive, Ki = 0.29 microM (competitive, Ki = 0.23 microM). A range of detergents solubilized and activated microsomal chitinase in a highly specific manner. Following density gradient centrifugation of microsomes, chitinase was distributed approximately evenly throughout the gradient suggesting that microsomal chitinase is not associated exclusively with any one membrane component. The possible morphogenetic role of microsomal chitinase is discussed in relation to the potential of this enzyme as a target for highly specific antifungal agents.