Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein

cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.     

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 of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

Early Secretory Pathway Gene TRS85 is Required for Selective Macroautophagy of Peroxisomes in Yarrowia lipolytica

Yarrowia lipolytica was recently introduced as a new model organism to study peroxisome degradation in yeasts. Transfer of Y. lipolytica cells from oleate/ethylamine to glucose/ammonium chloride medium leads to selective macroautophagy of peroxisomes. To decipher the molecular mechanisms of macropexophagy we made use of Y. lipolytica tagged mutants affected in the inactivation of peroxisomal enzymes under pexophagy conditions, Ain16 and Ain19. Both strains appeared to be disrupted at two different sites of the same gene, YlTRS85, the ortholog of Saccharomyces cerevisiae TRS85 that encodes 85 kDa subunit of transport protein particle (TRAPP). Y. lipolytica trs85 mutants had multiple defects of protein transport to external medium, cell wall and vacuoles, indicating that YlTrs85 is indeed the ScTrs85 functional homologue, required early in the classical secretory pathway. Interestingly, peroxisomes were not able to reach vacuoles under pexophagy conditions in both Ain16 and Ain19 strains. Therefore, the essential role of the early secretory flow in selective macroautophagy of peroxisomes is suggested.     

Flagellar proteins of motile spores ofActinomycetes

Flagella of some of the actinoplanete genera were purified and the molecular sizes of their flagellin subunits compared by SDS-PAGE analysis to flagellins of cells of other bacteria. Several species ofActinoplanes have a major flagellar protein of subunit sizes of 42–43 kDa and a lesser amount of a second protein, possibly a minor flagellin subunit, of 60 kDa. The flagellar protein sizes of other actinoplanetes ranged from 32–43 kDa (major) and 48–58 kDa (minor). Antibodies formed against the 42-kDa protein ofA. rectilineatus showed cross-reactivity in Western blots against flagellar proteins of spores of otherActinoplanes species, two species ofDactylosporangium and anAmpullariella species. Cross-reactivity was also observed with motile cells of two other actinomycetes,Arthrobacter atrocyaneus and aGeodermatophilus species, and withBacillus subtilis. No cross-reactivity was observed withEscherichia coli orPlanomonospora parontospora flagellar proteins. The amino acid composition and partial N-terminal sequence of the 42-kDa flagellar protein ofA. rectilineatus was compared to literature data for other bacterial flagellins and found to be most similar toB. subtilis 168.     

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5′-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5′-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.     

A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes

Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN‐) and high resolution clear native (hrCN‐) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine‐ and deoxycholate‐based native (HDN‐) PAGE. We compared the capacity of HDN‐, BN‐ and hrCN‐PAGE to resolve the well‐studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN‐PAGE. The analysis of isolated chloroplast envelope complexes by HDN‐PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN‐PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.     

SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52

The Schizosaccharomyces pombe rad31 and hus5 genes are required for the DNA damage response, as mutants defective in these genes are sensitive to DNA damaging agents, such as UV and ionising radiation and to the DNA synthesis inhibitor hydroxyurea (HU). Sequence analysis has suggested that rad31 and hus5 encode components of the Pmt3 (SUMO) modification process in S.pombe. We show here that the rad31 null and hus5.62 mutants display reduced levels of Pmt3 modification. We have initiated a search for proteins required for the DNA damage response, which may be modified by Pmt3 and have identified Rad22, the fission yeast homologue of the recombination protein Rad52. Purification of myc + His-tagged Rad22 protein from cells expressing HA-tagged Pmt3 identifies an 83 kDa species which cross-reacts with anti-HA antisera. We show here that Rad22 interacts with Rhp51 and Rpa70 (the fission yeast homologues of Rad51 and the large subunit of RPA, respectively), but that neither of these proteins appears to be responsible for the 83 kDa species. The 83 kDa species is observed when extracts are prepared under both native and denaturing conditions, and is also observed when myc + His-tagged Rad22 and Pmt3 are expressed at wild type levels, suggesting that Rad22 is modified by Pmt3 in vivo. We have established an S.pombe in vitro Pmt3 modification system and have shown that Rad22 and Rhp51 are modified in vitro, but that Rpa70 is not.     

代谢工程改造解脂耶氏酵母合成羧酸的研究进展

解脂耶氏酵母是一种具有独特生理代谢特征的非常规酵母.它具有可以利用多种廉价碳源、低pH值耐受性好、分泌能力强等优点,因此非常适合用于各种工业产品的微生物发酵.目前,解脂耶氏酵母已被证实具有高效生产多种(同源或异源)有机羧酸的能力.本文对近年来利用代谢工程及合成生物学技术改造解脂耶氏酵母生产羧酸的实例进行了总结,并重点介...     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition paericle

Erratum

The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition paericle     

Identification of Candida isolated from the cutaneous candidiasis by the combined use of confirmatory medium and slide agglutination with monofactorial antibodies

Forty strains ofCandida and one ofTorulopsis were isolated from patients with cutaneous candidiasis. The isolates comprised 29 strains ofC. albicans, 7 strains ofC. tropicalis, 2 strains ofC. guilliermondii, and one each ofC. parakrusei, C. lipolytica, andT. famata were identified by the ordinary method. Besides the common pathogenC. albicans, a few other species ofCandida may be etiologic organisms of cutaneous candidiasis. These strains were re-examined by combined use of sucrose agar slants and slide agglutination tests with IgG monofactorial antibodies as a rapid identification method, especially for determining serotypes ofC. albicans. The new method was useful and reliable for rapid identification ofC. albicans and related species. All strains ofC. albicans isolated from skin lesions proved to be standard serotypes ofC. albicans.

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Zusammenfassung Vierzig Stämme vonCandida und eins vonTorulopsis wurden aus Kranken mit kutanen Candidamykosen isoliert. Neunundzwanzig Stämme vonC. albicans, 7 vonC. tropicalis, 2 vonC. guilliermondii, und je einer vonC. parakrusei, C. lipolytica undT. famata wurden mit dem ordinären Methode identifiziert. Außer dem gewohnlichen Erreger,C. albicans, konnten auch ein Paar andere Spezies vonCandida als den Erreger betrachtet werden. Sechsunddreißig Stämme vonC. albicans undC. tropicalis wurden mit der von uns verbesserten kombinierten serologischen und biologischen Methode untersucht, besonders um den Serotypus vonC. albicans festzusetzen. Die neue Methode war gut und zuverlässig als die rapide Identification vonC. albicans und verwandten Spezies. Alle aus der Hautläsion isoliertenC. albicans waren der in Japan allgemeine Serotypus vonC. albicans.

     

Isolation and cloning of the Yarrowia lipolytica SEC65 gene,a component of the yeast signal recognition particle displaying homology with the human SRP19 gene

The signal recognition particle (SRP) is a ribonucleoprotein composed of a 7SL RNA and six polypeptides. Here we report the results of a series of experiments carried out to define the function of the Yarrowia lipolytica homologue of the 19 kDa subunit of mammalian SRP. The YlSEC65 gene product is a 310 amino acid protein. Coimmuneprecipitation of Sec65p and 7SL RNA in Y. lipolytica revealed that these components are stable associated in a complex. Deletion of the YlSEC65 gene is lethal, in contrast with the results described for the Saccharomyces cerevisiae SEC65 gene, which is not essential for cell growth and whose deletion results in slowly growing strains. Using site-directed mutagenesis we demonstrate that the two arginine residues of the EGRR motif conserved in all SRP19 homologues are essential for SRP activity. By random mutagenesis of YlSEC65, we have isolated a temperature-sensitive mutant and shown that it was affected in protein secretion at the non-permissive temperature. We also show that the YlSEC65 gene is able to functionally complement the temperature-sensitive growth of S. cerevisiae sec65 mutants. Our results suggest that SRP-dependent targeting may be the main secretory pathway in Y. lipolytica, as has been described for higher eukaryotes.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

A sterol biosynthetic geneAtCYP51A2 promoter for constitutive and ectopic expression of a transgene in plants

Arabidopsis CYP51A2 (AtCYP51A2) mediates the sterol 14α-demethylation step inde novo sterol biosynthesis, and is constitutively and highly expressed in all plant tissues (Kim et al., 2005). We exploited the molecular features of its expression and the fundamental role of sterol biosynthesis in cells to develop a plant-derived promoter. Our GUS expression analysis between transgenicArabidopsis lines forAtCYP51A2::GUS and35S::GUS revealed that activity of theAtCYP51A2 promoter was comparable to that of the35S promoter, based on enzymatic activities and protein levels. TheAtCYP51A2 promoter was also constitutively active in transgenic tobacco, indicating that 5′ regulatory elements could be conserved amongCYP51 promoters in dicot plants. A homologue ofAtCYP51A2 was identified from rape seed, a crop species closely related toArabidopsis. Its constitutive tissue expression pattern implies that the application of thisAtCYP51A2 promoter is possible for that species. Based on these results, we present a new binary vector system with the plant-derivedAtCYP51A2 promoter, which is able to constitutively and ectopically drive a transgene in various dicotyledonous plants. These two authors are equally contributed to this work.     

Major sclerotial polypeptides of psychrophilic pathogenic fungi: Intracellular localization and antigenic relatedness

Summary Major sclerotial polypeptides from the psychrophiles,Myriosclerotinia borealis (W 51),Coprinus psychromorbidus (LRS 131),Typhula idahoensis (W 21), andTyphula incarnata (W 21) were purified by using polyacrylamide gel electrophoresis and electroelution. Polyclonal antibodies were raised against these major sclerotial polypeptides. Immunofluorescence microscopy showed that the major sclerotial polypeptides from all four psychrophilic species were sequestered in discrete protein bodies of cultured and field-grown sclerotia. Western blot analysis indicated that all antisera reacted positively with their respective antigens, the major sclerotial polypeptides. Reciprocal immunological cross-reactions were observed between the major sclerotial polypeptides ofM. borealis (W 51) andT. idahoensis (W 21). Antiserum to the major sclerotial polypeptides of bothM. borealis andT. idahoensis also recognized the major sclerotial polypeptides ofC. psychromorbidus (LRS 131). It is suggested that the major sclerotial polypeptides of these psychrophilic plant pathogens may act as storage proteins.Abbreviations W 51 Myriosclerotinia borealis (W 51) - LRS 131 Coprinus psychromorbidus (LRS 131) - W 21 Typhula idahoensis (W 21) - W 29 Typhula incarnata (W 29) - anti W 51 antiserum to the major sclerotial polypeptide ofM. borealis W 51 - anti LRS 131 antiserum to the major sclerotial polypeptides ofC. psychromorbidus (LRS 131) - anti W 21 antiserum to the major sclerotial polypeptides ofT. idahoensis (W 21) - anti W 29 antiserum to the major sclerotial polypeptides ofT. incarnata (W 29) - SDS sodium dodecylsulfate - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - HRP horseradish peroxidase - PBS phosphate buffered saline - TBS Tris buffered saline - FITC fluorescein isothiocyanate     

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.