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.

Structural and functional aspects of peroxisomal membranes in yeasts.

The peroxisomal membrane compartmentalizes specific metabolic functions in the intermediary metabolism of various aerobic eukarya. In yeast, peroxisomal membranes are typified by their small width (+/- 7-8 nm) and absence of large integral membrane proteins in freeze-etch replicas. They show a unique polypeptide profile which, in contrast to their phospholipid composition, differs from that of other membranes in the cell. Part of these proteins are substrate-inducible and are probably related to specific peroxisomal function(s). In vivo, the observed proton motive force across the peroxisomal membrane may play a role in the function of the organelle in that it contributes to the driving force required for selective transport of various enzyme substrates and/or metabolic intermediates. To date only few peroxisomal membrane proteins (PMPs) have been functionally characterized. A major constitutive 31-kDa PMP present in the peroxisomal membrane of Hansenula polymorpha has been purified and was shown to display pore-forming properties. In addition, a peroxisomal H(+)-ATPase has been identified which most probably is involved in the generation/maintenance of the in vivo pH gradient across the peroxisomal membrane. Other functions of peroxisomal membrane proteins remain obscure although the first genes encoding yeast PMPs are now being cloned and sequenced. Studies on peroxisome-deficient yeast mutants revealed that specific peroxisome functions are strictly dependent on the intactness of the peroxisomal membrane. In this contribution several examples are presented of metabolic disorders due to peroxisomal malfunction in yeast.

The in vitro permeability of yeast peroxisomal membranes is caused by a 31 kDa integral membrane protein.

A major 31 kDa integral peroxisomal membrane protein (PMP31) of Hansenula polymorpha was purified to homogeneity from isolated peroxisomal membranes by FPLC after solubilization by Triton X-100. Biochemical analysis indicated that this protein, which showed cross-reactivity with antibodies against the 31 kDa porin of the mitochondrial outer membrane of Saccharomyces cerevisiae, had pore-forming properties. Firstly, proteoliposomes composed of asolectin and purified PMP31 showed selective permeability, determined as the [14C]sucrose/[3H]dextran leakage ratios. Furthermore, the generation of a delta psi by potassium diffusion gradients was negatively affected by the presence of PMP31 in asolectin liposomes. A similar effect was observed in proteoliposomes containing purified cytochrome c oxidase as a delta psi generating system. Control experiments confirmed that the observed leakage is significant and introduced by the incorporation of PMP31 protein. Selective sucrose leakage was abolished in samples pretreated with glutaraldehyde; an identical effect of glutaraldehyde was, however, not observed for the membrane potential measurements.

Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha.

We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous culture on a glucose/methanol mixture, indicated that various PMPs were normally synthesized. As in wild type (WT) cells, the levels of PMP synthesis appeared to be dependent on specific cultivation conditions, e.g. the carbon source used for growth. In contrast to WT controls, PMPs in methanol-induced per mutants were not subject to proteolytic degradation. Biochemical and immuno(cyto)chemical studies suggested that the PMPs in methanol-induced per cells were located in small proteinaceous aggregates, separated from peroxisomal matrix proteins that were also present in the cytosol. Vesicular membranous structures, resembling the morphology of intact peroxisomes, were never detected irrespective of the growth conditions employed.

Expression and targeting of a 47 kDa integral peroxisomal membrane protein of Candida boidinii in wild type and a peroxisome-deficient mutant of Hansenula polymorpha.

A 47 kDa integral peroxisomal membrane protein (PMP47) of Candida boidinii was expressed in wild type (WT) and a temperature-sensitive (Ts6) peroxisome-deficient (per) mutant of Hansenula polymorpha. The subcellular location of PMP47 appeared to be dependent on the level of expression. At low expression levels PMP47 was sorted to the peroxisomal membrane; however, in Ts6 cells grown at restrictive temperatures (which lack intact peroxisomes) PMP47 was solely located in small cytosolic aggregates, together with homologous H. polymorpha PMP's. At enhanced expression levels, however, part of the protein also became incorporated into mitochondria, both in transformed WT and Ts6 cells.

Proliferation and metabolic significance of peroxisomes in Candida boidinii during growth on D-alanine or oleic acid as the sole carbon source.

We have studied the induction of peroxisomes in the methylotrophic yeast Candida boidinii by D-alanine and oleic acid. The organism was able to utilize each of these compounds as the sole carbon source and grew with growth rates of mu = 0.20 h-1 (on D-alanine) or mu = 0.43 h-1 (on oleic acid). Growth was associated with the development of many peroxisomes in the cells. On D-alanine a cluster of tightly interwoven organelles was observed which made up 6.3% of the cytoplasmic volume and were characterized by the presence of D-amino acid oxidase and catalase. On oleic acid rounded to elongated peroxisomes were dominant which were scattered throughout the cytoplasm. These organelles contained increased levels of beta-oxidation enzymes; their relative volume fraction amounted 12.8% of the cytoplasmic volume.

Permeability properties of peroxisomal membranes from yeasts.

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH1 complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence of peroxisomal membrane proteins in methylotrophic yeasts grown under different conditions.

We have studied the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained indicated that no significant ultrastructural differences existed between the membranes of variously grown cells. The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate-polyacrylamide gels of purified microbody membranes isolated from methanol-grown Hansenula polymorpha, prominent protein bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph, Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D-alanine instead of ammonium sulphate as the nitrogen source.

A proton-translocating adenosine triphosphatase is associated with the peroxisomal membrane of yeasts.

The association of an ATPase with the yeast peroxisomal membrane was established by both biochemical and cytochemical procedures. Peroxisomes were purified from protoplast homogenates of the methanol-grown yeast Hansenula polymorpha by differential and sucrose gradient centrifugation. Biochemical analysis revealed that ATPase activity was associated with the peroxisomal peak fractions which were identified on the basis of alcohol oxidase and catalase activity. The properties of this ATPase closely resembled those of the mitochondrial ATPase of this yeast. The enzyme was Mg2+-dependent, had a pH optimum of approximately 8.5 and was sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), oligomycin and azide, but not to vanadate. A major difference was the apparent Km for ATP which was 4-6 mM for the peroxisomal ATPase compared to 0.6-0.9 mM for the mitochondrial enzyme. Cytochemical experiments indicated that the peroxisomal ATPase was associated with the membranes surrounding these organelles. After incubations with CeCl3 and ATP specific reaction products were localized on the peroxisomal membrane, both when unfixed isolated peroxisomes or formaldehyde-fixed protoplasts were used. This staining was strictly ATP-dependent; in controls performed in the absence of substrate, in the presence of glycerol 2-phosphate instead of ATP, or in the presence of DCCD, staining was invariably absent. Similar staining patterns were observed in subcellular fractions and protoplasts of Candida utilis and Trichosporon cutaneum X4, grown in the presence of ethanol/ethylamine or ethylamine, respectively.