We have analyzed the functions of two vacuolar t-SNAREs, Vam3p and Vam7p, in peroxisome degradation in the methylotrophic yeast Hansenula polymorpha. A Hp-vam7 mutant was strongly affected in peroxisome degradation by selective macropexophagy as well as non-selective microautophagy. Deletion of Hp-Vam3p function had only a minor effect on peroxisome degradation processes. Both proteins were loc...

We have studied Hansenula polymorpha Pex5p and Pex20p, peroxins involved in peroxisomal matrix protein import. In vitro binding experiments suggested that H. polymorpha Pex5p and Pex20p physically interact. We used single particle electron microscopy (EM) to analyze the structure of purified Pex5p and its possible association with Pex20p. Upon addition of Pex20p, a multimeric Pex20p complex was...

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding alpha-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated alpha-COP lacking more than 300 C-terminal amino acids, manifes...

Previously we demonstrated that peroxisomicine A1 (T-514), a plant toxin isolated from Karwinskia species, has a deteriorating effect on the integrity of peroxisomes of methylotrophic yeasts. Here we describe two strains of Hansenula polymorpha, affected in the normal utilization of methanol as sole source of carbon and energy due to peroxisomicine A1 treatment. The two strains isolated (L17 an...

Saccharomyces cerevisiae is unable to grow on methanol because it lacks the enzymes required for its metabolism. To study the possibility of whether or not the methanol oxidation pathway of Hansenula polymorpha can be transferred to S. cerevisiae, the gene coding for alcohol oxidase, a peroxisomal homo-octameric flavoprotein, was introduced into S. cerevisiae. Transformed cells contain varying ...

Hansenula polymorpha Pex14p (HpPex14p) is a component of the peroxisomal membrane essential for peroxisome biogenesis. Here, we show that HpPex14p is phosphorylated in vivo. In wild-type H. polymorpha cells, grown in the presence of [32P]orthophosphate, the 32P label was incorporated into HpPex14p. Labelled HpPex14p was induced after a shift of cells to methanol-containing media and rapidly dis...

We have isolated the Penicillium chrysogenum pex5 gene encoding the receptor for microbody matrix proteins containing a type 1 peroxisomal targeting signal (PTS1). Pc-pex5 contains 2 introns and encodes a protein of approximately 75 kDa. P. chrysogenum pex5 disruptants appear to be highly unstable, show poor growth, and are unable to sporulate asexually. Furthermore, pex5 cells mislocalize a fl...

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 diss...

We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequen...