Vladimir I. Titorenko

The Hansenula polymorpha PDD1 gene product, essential for the selective degradation of peroxisomes, is a homologue of Saccharomyces cerevisiae Vps34p

Via functional complementation we have isolated the Hansenula polymorpha PDD1 gene essential for selective, macroautophagic peroxisome degradation. HpPDD1 encodes a 116u2009kDa protein with high similarity (42% identity) to Saccharomyces cerevisiae Vps34p, which has been implicated in vacuolar protein sorting and endocytosis. Western blotting experiments revealed that HpPDD1 is expressed constitutively. In a H. polymorpha pdd1 disruption strain peroxisome degradation is fully impaired. Sequestered peroxisomes, typical for the first stage of peroxisome degradation in H. polymorpha, were never observed, suggesting that HpPdd1p plays a role in the tagging of redundant peroxisomes and/or sequestration of these organelles from the cytosol. Possibly, HpPdd1p is the functional homologue of ScVps34p, because—like S. cerevisiae vps34 mutants—H. polymorpha pdd1 mutants are temperature‐sensitive for growth and are impaired in the sorting of vacuolar carboxypeptidase Y. Moreover, HpPdd1p is associated to membranes, as was also observed for ScVps34p. Copyright

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.

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.

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.