Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha Pex14 null mutant. (1/27)

Hansenula polymorpha Deltapex14 cells are affected in peroxisomal matrix protein import and lack normal peroxisomes. Instead, they contain peroxisomal membrane remnants, which harbor a very small amount of the major peroxisomal matrix enzymes alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS). The bulk of these proteins is, however, mislocated in the cytosol. Here, we show that in Deltapex14 cells overproduction of the PTS1 receptor, Pex5p, leads to enhanced import of the PTS1 proteins AO and DHAS but not of the PTS2 protein amine oxidase. The import of the PTS1 protein catalase (CAT) was not stimulated by Pex5p overproduction. The difference in import behavior of AO and CAT was not related to their PTS1, since green fluorescent protein fused to the PTS1 of either AO or CAT were both not imported in Deltapex14 cells overproducing Pex5p. When produced in a wild type control strain, both proteins were normally imported into peroxisomes. In Deltapex14 cells overproducing Pex5p, Pex5p had a dual location and was localized in the cytosol and bound to the outer surface of the peroxisomal membrane. Our results indicate that binding of Pex5p to the peroxisomal membrane and import of certain PTS1 proteins can proceed in the absence of Pex14p.  (+info)

Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation. (2/27)

Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non-covalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH-containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.  (+info)

Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii. (3/27)

We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.  (+info)

A methylotrophic pathway participates in pectin utilization by Candida boidinii. (4/27)

The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.  (+info)

Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments. (5/27)

Alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS) constitute the bulk of matrix proteins in methylotrophic yeasts, model organisms for the study of peroxisomal assembly. Both are homooligomers; AO is a flavin-containing octamer, whereas DHAS is a thiamine pyrophosphate-containing dimer. Experiments in recent years have demonstrated that assembly of peroxisomal oligomers can occur before import; indeed the absence of chaperones within the peroxisomal matrix calls into question the ability of this compartment to assemble proteins at all. We have taken a direct pulse-chase approach to monitor import and assembly of the two major proteins of peroxisomes in Candida boidinii. Oligomers of AO are not observed in the cytosol, consistent with the proteins inability to undergo piggyback import. Indeed, oligomerization of AO can be followed within the peroxisomal matrix, directly demonstrating the capacity of this compartment for protein assembly. By contrast, DHAS quickly dimerizes in the cytosol before import. Binding and import was slowed at 15 degrees C; the effect on AO was more dramatic. In conclusion, our data indicate that peroxisomes assemble AO in the matrix, while DHAS undergoes dimerization prior to import.  (+info)

The final acylation step in taxol biosynthesis: cloning of the taxoid C13-side-chain N-benzoyltransferase from Taxus. (6/27)

The formation of several acyl groups and an amide group of Taxol is catalyzed by regioselective CoA thioester-dependent acyltransferases. Several full-length acyltransferase sequences, obtained from a cDNA library constructed from mRNA isolated from Taxus cuspidata cells induced for Taxol production with methyl jasmonate, were individually expressed in Escherichia coli, from which a cDNA clone encoding a 3'-N-debenzoyl- 2'-deoxytaxol N-benzoyltransferase was identified. This recombinant enzyme catalyzes the stereoselective coupling of the surrogate substrate N-debenzoyl-(3'RS)-2'-deoxytaxol with benzoyl-CoA to form predominantly one 3'-epimer of 2'-deoxytaxol. The product 2'-deoxytaxol was confirmed by radio-HPLC,(1)H-NMR, and chemical ionization-MS. This enzymatic reaction constitutes the final acylation in the Taxol biosynthetic pathway. The full-length cDNA coding for the N-benzoyltransferase has an ORF of 1,323 nucleotides and encodes a 441-residue protein with a calculated molecular weight of 49,040. The recombinant enzyme expressed in E. coli has a pH optimum at 8.0, a k(cat) approximately 1.5 +/- 0.3 s(-1) and K(m) values of 0.42 mM and 0.40 mM for the N-deacylated taxoid and benzoyl-CoA, respectively. In addition to improving the production yields of Taxol in genetically engineered host systems, this enzyme provides a means of attaching modified aroyl groups to taxoid precursors for the purpose of improving drug efficacy.  (+info)

Import of assembled PTS1 proteins into peroxisomes of the yeast Hansenula polymorpha: yes and no! (7/27)

Previously, Waterham et al. [EMBO J. 12 (1993) 4785] reported that cytosolic oligomeric alcohol oxidase (AO) is not incorporated into peroxisomes after reassembly of the organelles in the temperature-sensitive peroxisome-deficient mutant pex1-6(ts) of Hansenula polymorpha shifted to permissive growth conditions. Here, we show that the failure to import assembled AO protein is not exemplary for other folded proteins because both an artificial peroxisomal matrix protein, PTS1-tagged GFP (GFP.SKL), and the endogenous dimeric PTS1 protein dihydroxyacetone synthase (DHAS) were imported under identical conditions. In vitro receptor-ligand binding studies using immobilised H. polymorpha Pex5p and crude extracts of methanol-induced pex1-6(ts) cells, showed that AO octamers did not interact with the recombinant PTS1 receptor, at conditions that allowed binding of folded GFP.SKL and dimeric DHAS. This shows that import of oligomeric proteins is not a universal pathway for peroxisomal matrix proteins.  (+info)

Transcriptional down-regulation of peroxisome numbers affects selective peroxisome degradation in Hansenula polymorpha. (8/27)

We have isolated and characterized a novel transcription factor of Hansenula polymorpha that is involved in the regulation of peroxisomal protein levels. This protein, designated Mpp1p, belongs to the family of Zn(II)2Cys6 proteins. In cells deleted for the function of Mpp1p the levels of various proteins involved in peroxisome biogenesis (peroxins) and function (enzymes) are reduced compared with wild type or, in the case of the matrix protein dihydroxyacetone synthase, fully absent. Also, upon induction of mpp1 cells on methanol, the number of peroxisomes was strongly reduced relative to wild type cells and generally amounted to one organelle per cell. Remarkably, this single organelle was not susceptible to selective peroxisome degradation (pexophagy) and remained unaffected during exposure of methanol-induced cells to excess glucose conditions. We show that this mechanism is a general phenomenon in H. polymorpha in the case of cells that contain only a single peroxisome.  (+info)

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