Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes

Abstract

Inactivation of peroxisomal enzymes in the yeast Hansenula polymorpha was studied following transfer of cells into cultivation media in which their activity was no longer required for growth. After transfer of methanol-grown cells into media containing glucose - a substrate that fully represses alcohol oxidase synthesis - the rapid inactivation of alcohol oxidase and catalase was paralleled by a disappearance of alcohol oxidase and catalase protein. The rate and extent of this inactivation was dependent upon conditions of cultivation of cells prior to their transfer. This carbon catabolite inactivation of alcohol oxidase was paralleled by degradation of peroxisomes which occurred by means of an autophagic process that was initiated by the formation of a number of electron-dense membranes around the organelles to be degraded. Sequestration was confined to peroxisomes; other cell-components such as ribosomes were absent in the sequestered cell compartment. Also, cytochemically, hydrolytic enzymes could not be demonstrated in these autophagosomes. The vacuole played a major role in the subsequent peroxisomal breakdown since it provided the enzymes required for proteolysis. Two basically similar mechanisms were observed with respect to the administration of vacuolar enzymes into the sequestered cell compartment. The first mechanism involved incorporation of a small vacuolar vesicle into the sequestered cell compartment. The delimiting membrane of this vacuolar vesicle subsequently disrupted, thereby exposing the contents of the sequestered cell compartment to vacuolar hydrolases which then degraded the peroxisomal proteins. The second mechanism, observed in cells which already contained one or more autophagic vacuoles, included fusion of the delimiting membranes of an autophagosome with the membrane surrounding an autophagic vacuole which led to migration of the peroxisome inside the latter organelle. Peroxisomes of methanol-grown H. polymorpha were degraded individually. In one cell 2 or 3 peroxisomes might be subject to degradation at the same time, but they were never observed together in one autophagosome. However, fusions of autophagic vacuoles in one cell were frequently observed. After inhibition of the cell's energy-metabolism by cyanide ions or during anaerobic incubations the formation of autophagosomes was prevented and degradation was not observed.

A 31P NMR study of the internal pH of yeast peroxisomes

“…Clearly, the major change in these spectra is the disappearance of the P~-peak at the low pH position of its titration curve. At the subcellular level these transfers are characterized by a rapid degradation of the large peroxisomes present in the cells (for details on the mechanism of peroxisomal degradation, see Veenhuis et al 1983). Morphometrical analysis of sections of the cells indicated that the peroxisomal volume fraction had decreased approximately 12-fold and amounted to 3.4% of the cytoplasmic volume after 4 h of incubation.…”

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“…In fact, this question can only be properly answered if information becomes available about the molecular machinery involved in the process. In this respect, yeast cells may become extremely useful, because autophagy in yeast is essentially similar to that in mammalian cells (Veenhuis et al, 1983;Baba et al, 1994) and allows the application of molecular genetic procedures in order to identify gene products involved in this process. Complementation studies with yeast mutants deficient in autophagy, obtained by screening on loss of cell viability during nitrogen starvation, have shown the participation of at least 15 different protein components (Tsukada & Ohsumi, 1993), and many more will be identified (Thumm et al, 1994).…”

Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha

“…The peroxisomes are degraded individually by a highly selective autophagic process, and the degradation process appeared to be identical under either of the above conditions. As an initial step, organelles to be degraded were sequestered from the cytosol by a number of membranous layers, closely surrounding the organelle, which most probably were derived from the endoplasmic reticulum; subsequently, hydrolytic enzymes required for the degradation of the microbody contents were supplied by fusion of part of the vacuole with the sequestered organelle (21,27,29,32). The degradation of peroxisomes in H. polymorpha is a rapid process; generally, the total turnover of a single organelle is accomplished within 20 to 45 min (29).…”