Grietje Sulter

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 μ=0.20 h-1 (on d-alanine) or μ=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 β-oxidation enzymes; their relative volume fraction amounted 12.8% of the cytoplasmic volume.

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 i) in the absence of substrate, ii) in the presence of glycerol 2-phosphate instead of ATP, or iii) 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.

Evidence for functional heterogeneity among microbodies in yeasts

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.

Acetylsalicylic Acid and Acetaminophen to Combat Elevated Body Temperature in Acute Ischemic Stroke

Background: Because elevated body temperature (BT) in acute ischemic stroke is associated with poor outcome, it is currently recommended to maintain normothermia with antipyretics. Whether this can be achieved by the administration of acetylsalicylic acid (ASA) or acetaminophen is uncertain. Methods: Over a period of 9 months, patients admitted with acute hemiparetic ischemic stroke were monitored for BT in the first 48 h. When BT rose >37.5°C, patients were treated with either ASA 500 mg intravenously or acetaminophen suppository 1,000 mg. When 6 h later the BT was still or again >37.5°C, patients were further treated with acetaminophen suppository 1,000 mg up to 4 times a day. Primary outcome measure was normothermia (BT ≤37.5°C) within respectively 1 and 3 h after intervention. Secondary outcome parameters were any decrease in BT and the mean decrease in BT. Results: Of a total of 132 patients, 63 (48%) developed a BT >37.5°C. The effects of 43 injections of ASA and 89 administrations of acetaminophen were studied. After 1 h, treatment with acetaminophen produced both significantly more reductions in BT and normothermia (respectively in 60 and 20%) than treatment with ASA (respectively in 37 and 5%). At 3 h, both interventions had a similar effect, with normothermia being achieved in only 37–38%. Fever (>38.0°C) and evidence of an infection were related to unresponsiveness to treatment. Conclusions: In the majority of patients with acute ischemic stroke, ASA and acetaminophen are insufficient for reducing an elevated BT to a state of normothermia.

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.

Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase

Glutathione‐dependent formaldehyde dehydrogenase (FLD) is a key enzyme required forthe catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris homologue, the H. polymorpha gene does not contain an intron. The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca. 41 kDa) with 82% identity to P. pastoris Fld1p, 76% identity to the FLD protein sequence from n‐alkane‐assimilating yeast Candida maltosa and 63–64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes. The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions. The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g. methylamine, is used as nitrogen source. These properties render the FLD1 promoter of high interest for heterologous gene expression. The availability of the H. polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter. The sequence has been deposited in the GenBank/NCBI data library under Accession No. AF364077. Copyright

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.

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 (RCLHI 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. Generation of a membrane potential (Δψ) was also not observed when RCLHI complex was introduced into vesicles of purified peroxisomal membranes. The significance of the observed permeability of isolated yeast peroxisomal membranes to small molecules with respect to current and future in vitro import studies is discussed.

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 PMPs. At enhanced expression levels, however, part of the protein also became incorporated into mitochondria, both in transformed WT and Ts6 cells.

Regulation of carbon metabolism in bacteria

Bacteria have different regulatory systems to control carbon metabolism, in a number of cases employ the phosphoenolpyruvate phosphotransferase system (PTS) is used, to tune the transport of carbohydrate and/or transcription of genes specifying catabolic enzymes. Although the physiological consequences of this regulation are similar in Gramnegative bacteria and Gram-positive bacteria, the mechanisms are entirely different. In the Gramnegative negative bacteria the state of phosphorylation of the glucose-specific PTS IIA protein plays a central role in inducer exclusion and cAMP synthesis, whereas regulatory processes are triggered by the phosphorylation state of HPr in Gram-positive bacteria. In both mechanisms the activity of target enzymes is modulated upon allosteric interaction with a particular form of IIAglc or HPr. In some Gram-positive bacteria, however, the regulation takes place via direct PEP-dependent enzyme I/HPr-mediated phosphorylation of a IIA-like domain of the target protein. The functional and structural (dis)similarities of this IIA-like domain and ILAglc are discussed in this paper.