Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β₂. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts. Received: 20 December1997 / Accepted: 19 June 1997     

Cloning and characterization of a cDNA encoding the beta subunit of human casein kinase II

Previous studies [Summercorn et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838; Klarlung & Czech (1988) J. Biol. Chem. 263, 15872-15875] have indicated that Balb/c 3T3 cells and 3T3-L1 adipocytes incubated with insulin show increased casein kinase II activity within minutes, implicating this serine/threonine kinase as an early step in an insulin signaling pathway. We recently reported the isolation of a cDNA encoding an alpha subunit of human casein kinase II [Meisner et al. (1989) Biochemistry 28, 4072-4076] as an initial step toward examining the regulation of this enzyme. We now describe a HepG2 cell casein kinase II beta subunit cDNA of 2.57 kb containing 96 bases of 5' untranslated sequence, 645 bases of open reading frame, and 1832 bases of 3' untranslated sequence with two polyadenylation consensus signal sequences and two poly(A) stretches. The open reading frame of the human beta subunit cDNA was 77% and 87% identical with the Drosophila sequence at the nucleotide and amino acid levels, respectively, and 99% identical with the bovine amino acid sequence. RNA analysis of HepG2 cell RNA utilizing HepG2 beta subunit cDNA fragments as probes revealed one major band migrating at 1.2 kb and two minor bands migrating at 3.0 and 4.2 kb. Results from DNA analysis of HepG2 genomic DNA, consistent with results utilizing Drosophila genomic DNA, suggest the presence of a single gene for the beta subunit of casein kinase II.     

Isolation, sequencing, and disruption of the CKA1 gene encoding the alpha subunit of yeast casein kinase II.

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which must be encoded by separate genes (R. Padmanabha and C. V. C. Glover, J. Biol. Chem. 262:1829-1835, 1987). The gene encoding the 42-kilodalton alpha subunit has been isolated by screening a yeast genomic library with oligonucleotide probes synthesized on the basis of the N-terminal amino acid sequence of the polypeptide. This gene (designated CKA1) contains an intron-free open reading frame of 372 amino acid residues. The deduced amino acid sequence is 67% identical to the alpha subunit of Drosophila melanogaster casein kinase II. The CKA1 gene product appears to be distantly related to other known protein kinases but exhibits highest similarity to the CDC28 gene product and its homolog in other species. Gene replacement techniques have been used to generate a null cka1 mutant allele. Haploid and diploid strains lacking a functional CKA1 gene appear to be phenotypically wild type, presumably because of the presence of the alpha' gene. Interestingly, the CKA1 gene appears to be single copy in the yeast genome; i.e., the alpha' gene, whose existence is known from biochemical studies and protein sequencing, cannot be detected by low-stringency hybridization.     

Genomic organization of the yeast Yarrowia lipolytica

We produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates. Received: 27 March 1997; in revised form: 8 July 1997 / Accepted: 9 July 1997     

Peroxisome biogenesis in the yeast Yarrowia lipolytica

Extensive perexisome proliferation during growth on oleic acid, combined with the availability of excellent genetic tools, makes the dimorphic yeast, Yarrowia lipolytica, a powerful model system to study the molecular mechanisms involved in peroxisome biogenesis. A combined genetic, biochemical, and morphological approach has revealed that the endoplasmic reticulum (ER) plays an essential role in the assembly of functional peroxisomes in this yeast. The trafficking of some membrane proteins to the peroxisomes occurs via the ER, results in their glyco-sylation in the ER lumen, does not involve transit through the Golgi, and requires the products of the SEC238, SRP54, PEX1, and PEX6 genes. The authors' data suggest a model for protein import into peroxisomes via two subpopulations of ER-derived vesicles that are distinct from secretory vesicles. A kinetic analysis of the trafficking of peroxisomal proteins in vivo has demonstrated that membrane and matrix proteins are initially targeted to multiple vesicular precursors that represent intermediates in the assembly pathway of peroxisomes. The authors have also recently identified a novel cytosolic chaperone, Pex20p, that assists in the oligomerization of thiolase in the cytosol and promotes its targeting to the peroxisome. These data provide the first evidence that a chaperone-assisted folding and oligomerization of thiolase in the cytosol is required for the import of this protein into the peroxisomal matrix.     

The isolation and characterization of the pyruvate kinase-encoding gene from the yeast Yarrowia lipolytica.

The dimorphic yeast, Yarrowia lipolytica, has been developed as a useful expression/secretion system for heterologous proteins such as chymosin and tissue plasminogen activator. To further develop this expression system, we have cloned the gene (PYK) encoding the highly expressed glycolytic enzyme, pyruvate kinase (PYK). Genomic clones were selected by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that were conserved through evolution. The clones identified by hybridization contained overlapping DNA inserts. We have confirmed the identity of the cloned gene based on two criteria: (1) the nucleotide sequence of the proposed PYK gene predicts a protein that is highly homologous to the corresponding Saccharomyces cerevisiae enzyme, and (2) PYK-specific activity was increased twofold when wild-type Y. lipolytica strains were transformed with the isolated DNA. Interestingly, we found that the open reading frame of the Y. lipolytica PYK gene was interrupted by an intron. This represents the first report of an intron in a Y. lipolytica gene.     

Double-stranded RNA from a marine alkane-degrading yeast Yarrowia lipolytica

A strain of Yarrowia lipolytica isolated from a tropical estuarine environment showed the presence of a double-stranded RNA. The double-stranded RNA had a molecular size of 5kb and a G+C content of 61 mole%. The strain was not a killer. No specific physiological function could be assigned to this plasmid.     

Double-stranded RNA from a marine alkane-degrading yeast Yarrowia lipolytica

A strain of Yarrowia lipolytica isolated from a tropical estuarine environment showed the presence of a double-stranded RNA. The double-stranded RNA had a molecular size of 5kb and a G+C content of 61 mole%. The strain was not a killer. No specific physiological function could be assigned to this plasmid.     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae

Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.     

Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica

Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.     

Isocitric acid production from rapeseed oil by Yarrowia lipolytica yeast

Production of d _S-threo-isocitric acid (ICA) by yeast meets serious difficulties since it is accompanied by a simultaneous production of citric acid (CA) in significant amounts that reduces the yield of desired product. In order to develop an effective process of ICA production, 60 yeast strains of different genera (Candida, Pichia, Saccharomyces, Torulopsis, and Yarrowia) were tested for their ability to produce ICA from rapeseed oil; as a result, wild-type strain Yarrowia lipolytica VKM Y-2373 and its mutant Y. lipolytica 704-UV4-A/NG50 were selected as promising ICA producers. The effects of temperature, pH, aeration, and concentrations of rapeseed oil, iron, and itaconic acid on ICA production by selected strains were studied. Under optimal conditions (pH 6.0; aeration 50–55 %; rapeseed oil concentration of 20–60 gl^?1, iron ion concentration of 1.2 mg l^?1, and itaconic acid amount of 30 mM), selected strains of Y. lipolytica produced predominantly ICA with a low amount of a by-product, CA.     

Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica.

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of 297 amino acids with a relative molecular weight of 30,559. The gene codes for a putative 22-amino-acid prepeptide (signal sequence) followed by an additional 135-amino-acid propeptide containing a possible N-linked glycosylation site and two Lys-Arg peptidase-processing sites. The final Lys-Arg site occurs at the junction with the mature, extracellular form. The mature protease contains two potential glycosylation sites. AEP is a member of the subtilisin family of serine proteases, with 42.6% homology to the fungal proteinase K. The functional promoter is more than 700 base pairs long, allowing for the observed complex regulation of this gene. The 5' and 3' flanking regions of the XPR2 gene have structural features in common with other yeast genes.