Abstract. Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxis0mes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.

We have isolated a collection of peroxisome degradation-deficient (Pdd ؊ ) mutants of the yeast Hansenula polymorpha which are impaired in the selective autophagy of alcohol oxidase-containing peroxisomes. Two genes, designated PDD1 and PDD2, have been identified by complementation and linkage analyses. In both mutant strains, the glucose-induced proteolytic turnover of peroxisomes is fully prevented. The pdd1 and pdd2 mutant phenotypes were caused by recessive monogenic mutations. Mutations mapped in the PDD1 gene appeared to affect the initial step of peroxisome degradation, namely, sequestration of the organelle to be degraded by membrane multilayers. Thus, Pdd1p may be involved in the initial signalling events which determine which peroxisome will be degraded. The product of the PDD2 gene appeared to be essential for mediating the second step in selective peroxisome degradation, namely, fusion and subsequent uptake of the sequestered organelles into the vacuole. pdd1 and pdd2 mutations showed genetic interactions which suggested that the corresponding gene products may physically or functionally interact with each other.In yeasts, peroxisomes develop in response to metabolic needs (23,28). In Hansenula polymorpha, the organelles are strongly induced by methanol and, to a lesser extent, by a number of unusual nitrogen sources, including primary amines. The opposite may also occur: under certain conditions, peroxisomes may be actively degraded after a shift of cells to a new environment in which the organelles become redundant for growth (32).At present, two conditions which lead to a rapid turnover of alcohol oxidase-containing peroxisomes in H. polymorpha are known, namely, a shift of cells from methylotrophic to nonmethylotrophic conditions (29, 32) and irreversible inactivation of the organellar function (21, 27).The degradation process appeared to be energy dependent (29). 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). However, not all organelles of the peroxisomal population present in one cell are affected: in particular, the large mature peroxisomes are rapidly degraded. These and other results (26) strongly suggest that the organelles destined for degradation are specifically tagged. Recently, we have obtained indirect evidence that the signals, initiating peroxisome turnover, are not directed against the matrix proteins but inst...

We have studied the response of two methylotrophic yeasts (Hansenula polymorpha and Candida boidinii) to toxin T-514, a toxin lethal to man, extracted from the shrub Karwinska homboldtiana. Growth experiments indicated a doseresponse effect; at enhanced concentrations (50 /zg/ml) the different subcellular organelles rapidly disintegrated resulting in death of the cultures. At non-lethal concentrations (< 2 /zg/ml) growth ceased initially, but resumed after a lag period of 4 h. At the subcellular level a specific effect was observed on peroxisomal integrity. Distinct holes appeared in the peroxisoreal membranes, resulting in leakage of matrix proteins from these organeiles. In addition, import of newly synthesized proteins appeared to he

Harder, W., & Veenhuis, M. (1992). Studies on the effect of toxin T-514 on the integrity of peroxisomes in methylotrophic yeasts. FEMS Microbiology Letters, 91(3).

We have studied the intraperoxisomal location of catalase in peroxisomes of methanol‐grown Hansenula polymorpha by (immuno)cytochemical means. In completely crystalline peroxisomes, in which the crystalline matrix is composed of octameric alcohol oxidase (AO) molecules, most of the catalase protein is located in a narrow zone between the crystalloid and the peroxisomal membrane. In non‐crystalline organelles the enzyme was present throughout the peroxisomal matrix. Other peroxisomal matrix enzymes studied for comparison, namely dihydroxyacetone synthase, amine oxidase and malate synthase, all were present throughout the AO crystalloid. The advantage of location of catalase at the edges of the AO crystalloids for growth of the organism on methanol is discussed.