Melchior E. Evers

Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared from methanol-grown cells, DHAS cosedimented with the peroxisomal enzymes alcohol oxidase and catalase. Electron microscopy of this fraction showed that it contained mainly intact peroxisomes, whereas SDS-polyacrylamide gel electrophoresis revealed two major protein bands (75 and 78 kDa) which were identified as alcohol oxidase and DHAS, respectively. The localization of DHAS in peroxisomes was further established by immunocytochemistry. After immuno-gold staining carried out on ultrathin sections of methanol-grown H. polymorpha using DHAS-specific antibodies, labelling was confined to the peroxisomal matrix.

Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha

We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf‐limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome‐deficient/Rf‐autotrophic double mutant (Δper1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of Δper1.rif1 cells grown under Rf‐limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild‐type and Δper1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in Δper1 cells but absent/very low in wild‐type and rif1 cells. In vivo only these FAD‐containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.

Characterization of peroxisome-deficient mutants of Hansenula polymorpha.

In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut-) mutants are affected in genes required for peroxisome biogenesis (PER genes). Previously, we reported that one group of per mutants, termed Pim-, are characterized by the presence of a few small peroxisomes with the bulk of peroxisomal enzymes located in the cytosol. Here, we describe a second major group of per mutants that were observed to be devoid of any peroxisome-like structure (Per-). In each Per- mutant, the peroxisomal methanol-pathway enzymes alcohol oxidase, catalase and dihydroxyacetone synthase were present and active but located in the cytosol. Together, the Pim- and Per- mutant collections involved mutations in 14 different PER genes. Two of the genes, PER5 and PER7, were represented by both dominant-negative and recessive alleles. Diploids resulting from crosses of dominant per strains and wild-type H. polymorpha were Mut- and harbored peroxisomes with abnormal morphology. This is the first report of dominant-negative mutations affecting peroxisome biogenesis.

In vitro dissociation and re-assembly of peroxisomal alcohol oxidases of Hansenula polymorpha and Pichia pastoris

We have studied the in vitro inactivation/dissociation and subsequent reactivation/re‐assembly of peroxisomal alcohol oxidases (AO) from the yeasts Hansenula polymorpha and Pichia pastoris. Both proteins are homo‐oligomers consisting of eight identical subunits, each containing one FAD as the prosthetic group. They were both rapidly inactivated upon incubation in 80% glycerol, due to their dissociation into the constituting subunits, which however still contained FAD. Dilution of dissociated AO in neutral buffer lead to reactivation of the protein due to AO re‐assembly, as was demonstrated by non‐denaturing PAGE. After use of mixtures of purified AO from H. polymorpha and P. pastoris active hybrid AO oligomers were formed. When prior to dissociation FAD was chemically removed from AO, reactivation or re‐assembly did not occur independent of externally added FAD.

Affinity purification of molecular chaperones of the yeast Hansenula polymorpha using immobilized denatured alcohol oxidase

We used peroxisomal alcohol oxidase (AO) for the affinity purification of molecular chaperones from yeasts. Methodical studies showed that up to 0.8 mg of purified bacterial GroEL was able to bind per ml of immobilized denatured AO column material. Using crude extracts of Hansenula polymorpha or Saccharomyces cerevisiae, several proteins were specifically eluted with Mg‐ATP which were recognized by antibodies against hsp60 or hsp70. One H. polymorpha 70 kDa protein was strongly induced during growth at elevated temperatures, whereas a second 70 kDa protein as well as a 60 kDa protein showed strong protein sequence homology to mitochondrial SSCI and hsp60, respectively, from S. cerevisiae.

Uptake of the β-Lactam Precursor α-Aminoadipic Acid in Penicillium chrysogenum Is Mediated by the Acidic and the General Amino Acid Permease

ABSTRACT External addition of the β-lactam precursor α-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular α-aminoadipic acid concentration and an increase in penicillin production. The exact route for α-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of α-aminoadipic acid, although PcGap1 showed a higher affinity for α-aminoadipic acid than PcDip5 (Km values, 230 and 800 μM, respectively). Leucine strongly inhibits α-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the α-aminoadipic acid flux in β-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the α-aminoadipic acid flux.

Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family.

We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was constitutively present. Peptides derived from both Hsp72 and Hsc74 showed sequence homology to the cytosolic Saccharomyces cerevisiae Hsp70s, Ssa1p and Ssa2p. The gene encoding Hsp72 (designated HSA1) was cloned, sequenced and used to construct HSA1 disruption and HSA1 overexpression strains. Comparison of the stress tolerances of these strains with those of wild‐type H. polymorpha revealed that HSA1 overexpression negatively affected the tolerance of the cells to killing effects of temperature or ethanol, but enhanced the tolerance to copper and cadmium. The tolerance for other chemicals (arsenite, arsenate, H2O2) or to high osmolarity was unaffected by either deletion or overexpression of HSA1. The nucleotide sequence of HSA1 was submitted to the EMBL data library and given the Accession Number Z29379.

Cloning and characterization of an aromatic amino acid and leucine permease of Penicillium chrysogenum

The gene encoding the amino acid permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid permeases. The cDNA clone was used for expression of the permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid permease Gap1. Upon overexpression, an increase in the uptake of L-tyrosine, L-phenylalanine, L-tryptophan and L-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched beta-carbon atom.

FORMATION AND QUANTIFICATION OF PROTEIN COMPLEXES BETWEEN PEROXISOMAL ALCOHOL OXIDASE AND GROEL

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition or GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel‐shift assay using non‐denaturing polyacrylamide gel electrophoresis, and quantified by laser‐densitometry of the gels. In the presence of MgAMP‐PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition or GroFS.