As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol‐utilization‐defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase. Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol‐ and ethanol utilizing‐deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome‐deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol. The mutations complemented and therefore were recessive mutations in different genes.

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein(s) domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). C-terminal truncation mutants were constructed and expressed in Escherichia coli and their properties were analyzed. Remarkably, the entire IIA domain (160 amino acids) could be deleted without significant effect on lactose-H+ symport and galactoside equilibrium exchange. The phosphoenolpyruvate:sugar phosphotransferase system (PTS) catalyzes phosphoryl transfer from phosphoenolpyruvate (PEP) to sugars (e.g., glucose) via a number of energycoupling proteins-i.e., enzyme I, heat-stable protein HPr, IIA, and IIB (1). In addition to catalyzing sugar transport, the PTS is involved in regulation of non-PTS transport, carbon and nitrogen metabolism, chemotaxis, and other processes (1-3).In the Gram-negative enteric bacteria, transport of sugars can be regulated at the level of the transport enzyme itself (inducer exclusion) but also at the level of protein expression (induction, catabolite repression) (1, 3). This dual regulation allows an instantaneous response of the organism to the presence or absence of a specific sugar and a slow response, which involves switching on/off the transcription of certain genes. The PTS has a central role in this regulation since the phosphorylation state of the phosphoryl transfer protein IIAGlc affects the activity of various non-PTS transport enzymes (inducer exclusion) as well as cAMP synthesis (catabolite repression). The phosphorylation state of IIAGlc is determined by the balance between phosphorylation via HPr-P and dephosphorylation via IICBGlc in the presence of substrate (e.g., glucose). The result of this regulation is that when Escherichia coli grows in the presence of glucose (PTS sugar) and a non-PTS sugar like lactose or melibiose, diauxic growth is observed with glucose being used first (1, 3).The involvement of IIAGlc or IIA-like proteins in PTSmediated regulation in nonenteric bacteria-e.g., Grampositives-is unclear. The surprising observation has been made, however, that a number of non-PTS sugar transport proteins have a C-terminal extension that is homologous to IIAGIc of E. coli (4). The best-characterized system of this family of transport proteins with a two-domain structure is the lactose transport protein (LacS) of Streptococcus thermophilus (4-8). The LacS protein catalyzes the uptake of galactosides in symport with a proton or exchanges lactose for intracellularly formed galactose (6, 7). The N-terminal (carrier) domain of LacS is typical for a polytopic membrane protein and is composed of 12 a-helical transmembrane segments; the Cterminal IIA domain is hydrophilic and '160 amino acids in size (4). It has been suggested that structurally and functionally distinct domains such as the carrier and IIA part of LacS are connected by interdomain structures (or Q-linkers) that allow the different domains to interact functionally (9).In the prese...

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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.

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.