Identification of intragenic mutations in the Hansenula polymorpha PEX6 gene that affect peroxisome biogenesis and methylotrophic growth

Two interacting AAA ATPases, Pex1p and Pex6p, are indispensable for peroxisome biogenesis in different organisms. Mutations affecting corresponding genes are the most common cause of the peroxisome biogenesis disorders in humans. By UV mutagenesis of the Hansenula polymorpha pex6 mutant, deficient in peroxisome biogenesis, we isolated a conditional cold-sensitive strain with restored ability to grow in methanol medium at 37 degrees C but not at 28 degrees C. Sequencing of the pex6 allele revealed a point mutation in the first AAA module of the PEX6 gene that leads to substitution of a conserved amino acid residue (G737E). An additional intragenic mutation identified in the cold-sensitive pex6 allele leads to a conserved amino acid substitution in the second AAA domain (R1000G). Electron microscopic analysis revealed restored peroxisomes in methanol-induced cold-sensitive pex6 cells at both permissive and restrictive temperatures. If separated, the secondary mutation did not affect methylotrophic growth. Our data suggest that H. polymorpha Pex6p may have a complex function in peroxisome biogenesis in which identified amino acid residues are involved.     

Plant catalase is imported into peroxisomes by Pex5p but is distinct from typical PTS1 import

We have previously demonstrated that the targeting signal ofpumpkin catalase, Cat1, is an internal PTS1 (peroxisomal targetingsignal 1)-like sequence, QKL, located at –13 to –11from the C-terminus, which is different from the typical PTS1SKL motif located in the C-terminus. Here we show that Cat1import into peroxisome is dependent on the cytosolic PTS receptor,Pex5p, in Arabidopsis, similar to typical PTS1 import, and thatother components for transport of peroxisomal matrix proteinssuch as Pex14p, Pex13p, Pex12p and Pex10p also contribute tothe import of Cat1. Interestingly, however, we found that Cat1interacts with the N-terminal domain of Pex5p, but not the C-terminaldomain for interaction with the typical PTS1, revealing thatPex5p recognizes Cat1 in a manner distinct from typical PTS1.     

酵母过氧化物体生物合成缺陷突变株的诱变、筛选和鉴定

过氧化物体对生物的生长和发育非常重要,人类很多疾病就是由于过氧化物体生物合成缺陷引起。以解脂耶氏酵母E122为出发菌,采用硫酸二乙酯诱变,获得了两株过氧化物体生物合成缺陷突变株,其中一株为温度敏感的突变株。在正常生长条件下,突变株的免疫荧光分析显示弥散的染色模式,且在电镜下观察不到过氧化物体的形态结构。将克隆于表达载体pINA445上的目前所发现的与过氧化物体生物合成有关的基因转化这两株突变株,发现它们均不能恢复其在含油酸的培养基上的生长,表明这两个突变株是由与过氧化物体生物合成相关的新基因的突变引起。这两个突变株的获得为参与过氧化物体生物合成的新基因的发现奠定了基础。     

Proteomic analysis of highly purified peroxisomes from etiolated soybean cotyledons

To identify previously unknown peroxisomal proteins, we establishedan optimized method for isolating highly purified peroxisomesfrom etiolated soybean cotyledons using Percoll density gradientcentrifugation followed by iodixanol density gradient centrifugation.Proteins in highly purified peroxisomes were separated by two-dimensionalPAGE. We performed peptide mass fingerprinting of proteins separatedin the gel with matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry and used the peptide mass fingerprintsto search a non-redundant soybean expressed sequence tag database.We succeeded in assigning 92 proteins to 70 sequences in thedatabase. Among them, proteins encoded by 30 sequences werejudged to be located in peroxisomes. These included enzymesfor fatty acid β-oxidation, the glyoxylate cycle, photorespiratoryglycolate metabolism, stress response and metabolite transport.We also show experimental evidence that plant peroxisomes containa short-chain dehydrogenase/reductase family protein, enoyl-CoAhydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-likeprotein and a voltage-dependent anion-selective channel protein.     

Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.     

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes

 The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L. Received: 19 July 1999 / Accepted: 19 February 2000     

Potential role of annexin AnnAt1 from Arabidopsis thaliana in pH-mediated cellular response to environmental stimuli

Plant annexins, Ca(2+)- and membrane-binding proteins, are probably implicated in the cellular response to stress resulting from acidification of cytosol. To understand how annexins can contribute to cellular ion homeostasis, we investigated the pH-induced changes in the structure and function of recombinant annexin AnnAt1 from Arabidopsis thaliana. The decrease of pH from 7.0 to 5.8 reduced the time of the formation of ion channels by AnnAt1 in artificial lipid membranes from 3.5 h to 15-20 min and increased their unitary conductance from 32 to 63 pS. These changes were accompanied by an increase in AnnAt1 hydrophobicity as revealed by hydrophobicity predictions, by an increase in fluorescence of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) bound to AnnAt1 and fluorescence resonance energy transfer from AnnAt1 tryptophan residues to TNS. Concomitant lipid partition of AnnAt1 at acidic pH resulted in its partial protection from proteolytic digestion. Secondary structures of AnnAt1 determined by circular dichroism and infrared spectroscopy were also affected by lowering the pH from 7.2 to 5.2. These changes were characterized by an increase in beta-sheet content at the expense of alpha-helical structures, and were accompanied by reversible formation of AnnAt1 oligomers as probed by ultracentrifugation in a sucrose gradient. A further decrease of pH from 5.2 to 4.5 or lower led to the formation of irreversible aggregates and loss of AnnAt1 ionic conductance. Our findings suggest that AnnAt1 can sense changes of the pH milieu over the pH range from 7 to 5 and respond by changes in ion channel conductance, hydrophobicity, secondary structure of the protein and formation of oligomers. Further acidification irreversibly inactivated AnnAt1. We suggest that the pH-sensitive ion channel activity of AnnAt1 may play a role in intracellular ion homeostasis.     

Plant Peroxisomes: Molecular Basis of the Regulation of their Functions

β -oxidation, the glyoxylate cycle and the glycolate pathway for photorespiration. Recent molecular biological studies have revealed that most of these enzymes possess either one of two peroxisomal targeting signals (PTS) within their amino acid sequence. One of the signals, PTS1, is found at the carboxy-terminus, while the other, PTS2, is found within the amino-terminal presequence. Subsequent to the synthesis and folding of these enzymes in the cytosol, the targeting signal in the folded proteins may bind to the corresponding receptors. At present, only a receptor that recognizes PTS1 has been identified in higher plants. After the binding of the protein and the receptor, the protein complex may be recognized by docking proteins that exist in the peroxisomal membrane. The mechanisms responsible for the recognition of peroxisomal proteins are now under investigation. Genetic analyses of Arabidopsis mutants with defective peroxisomes may give us some clues to understanding the mechanisms of peroxisomal protein import. Received 18 November 1999/ Accepted in revised form 13 January 2000     

Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 × 10⁷  viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole^–) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole^– mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim^– (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 ×g supernatant, but produced wild-type levels of penicillin. Received: 16 April 1997 / Accepted: 26 July 1997     

Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves

Summary.  The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b ₅₆₁ proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b ₅₆₁ located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 °C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b ₅₆₁ proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24–31 kDa). Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary.     

New Functions of a Well-Known Protein: Prothymosin α Is Involved in Protecting Cells from Apoptosis and Oxidative Stress

Recent studies have revealed two new functions of prothymosin α (ProTα), a well-known protein and a subject of intense research. In addition to acting as an immunomodulator and stimulating cell proliferation, ProTα is involved in protecting the cell from apoptosis and regulating the expression of oxidative stress defense genes. The review considers the methods and approaches used to demonstrate the two new functions of ProTα.     

Molecular cloning and functional characterization of an endogenous endoglucanase belonging to GHF45 from the western corn rootworm, Diabrotica virgifera virgifera

A novel insect β-1,4-endoglucanase (DvvENGaseI) gene belonging to the glycoside hydrolase family (GHF) 45 was identified from the western corn rootworm, Diabrotica virgifera virgifera. The cDNA of the DvvENGaseI consisted of a 720 bp open reading frame encoding a 239 amino-acid protein. Analysis of the amino acid sequence revealed that DvvENGaseI exhibits 60% protein sequence identity when compared with an endoglucanase belonging to GHF45 from another beetle, Leptinotarsa decemlineata. Western blot analyses using a polyclonal antiserum developed from a partial peptide sequence revealed that DvvENGaseI expression coincided with body regions corresponding to the fore-, mid- and hindgut, although regions corresponding to the midgut and hindgut were the primary sites for DvvENGaseI expression. Functional analysis of the DvvENGaseI by RNA interference (RNAi) indicated that nearly complete knock-down of gene expression could be obtained by injection of dsRNA based on qRT-PCR and western blot analysis. However, suppression only resulted in slight developmental delays suggesting that this gene may be part of a larger system of cellulose degrading enzymes.