3 research outputs found

    Old yellow enzyme confers resistance of Hansenula polymorpha towards allyl alcohol

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    In the methylotrophic yeast, Hansenula polymorpha, peroxisomes are formed during growth on methanol as sole carbon and energy source and contain the key enzymes for its metabolism, one of the major enzymes being alcohol oxidase (AO). Upon a shift of these cells to glucose-containing medium, peroxisomes become redundant for growth and are rapidly degraded via a highly selective process designated macropexophagy. H. polymorpha pdd mutants are disturbed in macropexophagy and hence retain high levels of peroxisomal AO activity upon induction of this process. To enable efficient isolation of PDD genes via functional complementation, we make use of the fact that AO can convert allyl alcohol into the highly toxic compound acrolein. When allyl alcohol is added to cells under conditions that induce macropexophagy, pdd mutants die, whereas complemented pdd mutants and wild-type cells survive. Besides isolating bona fide PDD genes, we occasionally obtained pdd transformants that retained high levels of AO activity although their allyl alcohol sensitive phenotype was suppressed. These invariably contained extra copies of a gene cluster encoding omologues of Saccharomyces carlsbergensis old yellow enzyme. Our data suggest that the proteins encoded by these genes detoxify acrolein by converting it into less harmful components

    Hansenula polymorpha Vam7p is required for macropexophagy

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    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 located at the vacuolar membrane, with Hp-Vam7p also having a partially cytosolic location. Previously, in baker's yeast Vam3p and Vam7p have been demonstrated to be components of a t-SNARE complex essential for vacuole biogenesis. We speculate that the function of this complex in macropexophagy includes a role in membrane fusion processes between the outer membrane layer of sequestered peroxisomes and the vacuolar membrane. Our data suggest that Hp-Vam3p may be functionally redundant in peroxisome degradation. Remarkably, deletion of Hp-VAM7 also significantly affected peroxisome biogenesis and resulted in organelles with multiple, membrane-enclosed compartments. These morphological defects became first visible in cells that were in the mid-exponential growth phase of cultivation on methanol, and were correlated with accumulation of electron-dense extensions that were connected to mitochondria.
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