Sorting and function of peroxisomal membrane proteins.

Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells. Characteristic features of these organelles are their inducibility and their functional versatility. Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders. Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle. This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.

Transformation of carbon tetrachloride in an anaerobic packed-bed reactor without addition of another electron donor.

Carbon tetrachloride (52 microM) was biodegraded for more than 72% in an anaerobic packed-bed reactor without addition of an external electron donor. The chloride mass balance demonstrated that all carbon tetrachloride transformed was completely dechlorinated. Chloroform and dichloromethane were sometimes also found as transformation products, but neither accumulated to significant levels in comparison to the amount of carbon tetrachloride transformed. Transformation of carbon tetrachloride in the absence of an added electron donor suggests that carbon tetrachloride itself is the source of energy for the biological reaction observed, and possibly the source of carbon for cell growth. No such mechanism is yet known. The pathway of carbon tetrachloride transformation is not clear; it may be dehalogenated by hydrolytic reduction to carbon monoxide or formic acid which are electron demanding transformations. Carbon monoxide or formic acid may be further utilized and serve as electron donor. Complete dechlorination of carbon tetrachloride according to this pathway is independent of a second electron donor or electron acceptor, as with a fermentation process. Vancomycin, an inhibitor of gram positive eubacteria, severely inhibited carbon tetrachloride transformation in batch incubations with an enrichment culture from the reactor, indicating that gram positive eubacteria were involved in carbon tetrachloride transformation. Batch experiments with bromoethanesulfonic acid, used to inhibit methanogens, and molybdate, an inhibitor of sulfate reduction in sulfate reducing bacteria, demonstrated that neither methanogens nor sulfate reducers were involved in the complete dechlorination of carbon tetrachloride.

Thallium-201 uptake with negative iodine-131 scintigraphy and serum thyroglobulin in metastatic oxyphilic papillary thyroid carcinoma.

We report a case of a 48-yr-old woman who underwent surgery because of papillary oxyphilic thyroid carcinoma pT3. After total thyroidectomy, we administered 2960 MBq (131)I for ablation of the residual tissue. initial follow-up visits showed no clinical, radiological or scintigraphic evidence of residual or metastatic thyroid tissue. Serum thyroglobulin levels (Tg) and (131)I whole-body scintigraphy were negative. Three years after thyroidectomy, the patient experienced seizures, and as a consequence a brain tumor was removed. It was an undetected metastasis of the primary thyroid carcinoma. Histological examinations showed that neither the primary tumor nor the metastasis produced any Tg. With this fact in mind and the knowledge of negative (131)I whole-body scans we had to concentrate on radiological (CT and MRI scans) and nonspecific scintigraphic methods such as 201TI whole-body scintigraphy in our management of the patient. Further follow-up demonstrated multiple metastasis by 201TI whole-body scan (mediastinum, bones and soft tissue), and most of them have been removed by surgery. This case report demonstrates that, in addition to (131)I whole-body scans and measurement of serum Tg, the use of nonspecific tracers like 201TI is important to detect (131)I and/or Tg negative metastases.

Brefeldin A interferes with peroxisomal protein sorting in the yeast Hansenula polymorpha.

We have studied the effect of brefeldin A (BFA), a fungal toxin that interferes with coated vesicle formation, on the biogenesis of peroxisomes in the yeast Hansenula polymorpha. Addition of BFA (20 microg/ml) to cultures of H. polymorpha partially inhibited the development of peroxisomes and resulted in the reversible accumulation of newly synthesized peroxisomal membrane and matrix proteins at the endoplasmic reticulum. In contrast, BFA did not interfere with the selective degradation of peroxisomes. Taken together, our data suggest that the ER plays a crucial role in peroxisome biogenesis in H. polymorpha, possibly in the biosynthesis of the peroxisomal membrane.

Performance of a styrene-degrading biofilter containing the yeast Exophiala jeanselmei.

A general mathematical model developed for a description of pollutant degradation in a biofilm was used to evaluate the performance of a biofilter for the purification of styrene-containing gas. The biofilter contained perlite as an inert support on which a biofilm was present composed of a mixed microbial population containing the fungus Exophiala jeanselmei as a major styrene-degrading microorganism. Although styrene is a moderately hydrophobic compound, the biofilter was reaction limited at a styrene gas phase concentration of 0.1-2.4 g/m(3). Limitation of biofilter performance by the mass transfer of styrene was only observed at styrene concentrations lower than 0.06 g/m(3). A maximal styrene degradation rate of 62 g/(m(3). h) was maintained for over 1 year. At a high styrene concentration, the maximal styrene degradation rate could be increased to 91 g/(m(3). h) by increasing the oxygen concentration in the gas from 20 to 40%. After 300 days of operation, the dry-weight biomass concentration of the filter bed was 41% (w/w), and an average biofilm thickness of 240-280 microm, but maximal up to 600 microm, was observed. Experimental results and model calculations indicated an effective biofilm thickness of about 80 microm. It is postulated that the thickness of the effective biofilm is determined by the oxygen availability in the biofilm.

Transformation of carbon tetrachloride under sulfate reducing conditions.

The removal of carbon tetrachloride under sulfate reducing conditions was studied in an anaerobic packed-bed reactor. Carbon tetrachloride, up to a concentration of 30 microM, was completely converted. Chloroform and dichloromethane were the main transformation products, but part of the carbon tetrachloride was also completely dechlorinated to unknown products. Gram-positive sulfate-reducing bacteria were involved in the reductive dechlorination of carbon tetrachloride to chloroform and dichloromethane since both molybdate, an inhibitor of sulfate reduction, and vancomycin, an inhibitor of gram-positive bacteria completely inhibited carbon tetrachloride transformation. Carbon tetrachloride transformation by these bacteria was a cometabolic process and depended on the input of an electron donor and electron acceptor (sulfate). The rate of carbon tetrachloride transformation by sulfate reducing bacteria depended on the type of electron donor present. A transformation rate of 5.1 nmol x ml(-1) x h(-1) was found with ethanol as electron donor. At carbon tetrachloride concentrations higher than 18 microM, sulfate reduction and reductive dechlorination of carbon tetrachloride decreased and complete inhibition was observed at a carbon tetrachloride concentration of 56.6 microM. It is not clear what type of microorganisms were involved in the observed partial complete dechlorination of carbon tetrachloride. Sulfate reducing bacteria probably did not play a role since inhibition of these bacteria with molybdate had no effect on the complete dechlorination of carbon tetrachloride.

Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha.

We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (delta per1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of delta per1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and delta per1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in delta per1 cells but absent/very low in wild-type and rif1 cells. In vivo only these FAD-containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.

Styrene metabolism in Exophiala jeanselmei and involvement of a cytochrome P-450-dependent styrene monooxygenase.

The yeast-like fungus Exophiala jeanselmei degrades styrene via initial oxidation of the vinyl side chain to phenylacetic acid, which is subsequently hydroxylated to homogentisic acid. The initial reactions are catalyzed by a NADPH- and flavin adenine dinucleotide-dependent styrene monooxygenase, a styrene oxide isomerase, and a NAD(+)-dependent phenylacetaldehyde dehydrogenase. The reduced CO-difference spectrum of microsomal preparations of styrene-grown cells shows a characteristic absorption maximum at 450 nm, which strongly suggests the involvement of a cytochrome P-450-dependent styrene monooxygenase. Inhibition of styrene monooxygenase activity in cell extracts by cytochrome P-450 inhibitors SKF-525-A, metyrapone, and CO confirms this assumption.

Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides.

We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed under control of the H. polymorpha alcohol oxidase promoter (PAOX), were integrated into the genomic alcohol oxidase locus, yielding stable production strains. High-level synthesis of the fusion proteins, exceeding 20% of total cellular protein, was obtained when the transformed strains were grown in methanol-limited chemostat cultures; when expressed by itself, i.e. in the absence of the amine oxidase gene, IGF-II could not be recovered from crude cell extracts, probably as a result of rapid proteolytic degradation. Accumulation in peroxisomes did not significantly affect the IGF-II protein stability when expressed in the absence of the carrier protein. Apparently, fusion to the large (+/- 78 kDa) amine oxidase carrier particularly stabilizes the peptides and prevents them from proteolysis. After partial purification, the fusion partners were readily separated by factor Xa treatment.

Review: methylotrophic yeasts as factories for the production of foreign proteins.

In this contribution we discuss the potential of methylotrophic yeasts as hosts for the high level production of valuable foreign proteins. Recent relevant achievements on the intracellular production or secretion of proteins are summarized. Special attention is paid to a specific advantage of the use of methylotrophic yeasts, namely the possibility of accumulating the foreign gene products inside peroxisomes. This approach may be of major advantage when the protein product is toxic for the host cell and, also, to protect these proteins from undesired side-effects such as proteolysis or aggregation.

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources.

Expression of the peroxisome-deficient (Per-) phenotype by per mutants Hansenula of polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6ts and per14-11ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D-alanine (for both mutants) or methylamine (for per14-11ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno)cytochemical analyses indicated that during growth of cells on methanol in the presence of either D-alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.

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.

The Hansenula polymorpha PER3 gene is essential for the import of PTS1 proteins into the peroxisomal matrix.

PER genes are essential for the assembly of peroxisomes in Hansenula polymorpha. Here we describe the PER3 gene which was cloned by functional complementation of a H. polymorpha per3 mutant. The complementing PER3 gene encodes a protein of 569 amino acids (Per3p) with a calculated mass of 63.9 kDa; Per3p belongs to the tetratricopeptide repeat protein family and is located in both the cytosol and the peroxisomal matrix. Remarkably, Per3p does not contain a known targeting signal (PTS1 or PTS2). The PER3 gene product shows similarity to the Saccharomyces cerevisiae Pas10p (40% identity) and the Pichia pastoris Pas8p (55% identity). However, their function apparently cannot be interchanged since the P. pastoris PAS8 gene failed to functionally complement a H. polymorpha per3 disruption mutant. The per3 disruption mutant contained normal but small peroxisomes in which PTS2 proteins (both homologous and heterologous) were imported. Other matrix proteins (in particular PTS1 proteins) resided in the cytosol where they were normally assembled and active. We argue that Per3p is a component of the peroxisomal import machinery and most probably shuttles matrix proteins from the cytosol to the organellar matrix.

In vitro dissociation and re-assembly of peroxisomal alcohol oxidases of Hansenula polymorpha and Pichia pastoris.

We have studied the in vitro inactivation/dissociation and subsequent reactivation/re-assembly of peroxisomal alcohol oxidases (AO) from the yeasts Hansenula polymorpha and Pichia pastoris. Both proteins are homo-oligomers consisting of eight identical subunits, each containing one FAD as the prosthetic group. They were both rapidly inactivated upon incubation in 80% glycerol, due to their dissociation into the constituting subunits, which however still contained FAD. Dilution of dissociated AO in neutral buffer lead to reactivation of the protein due to AO re-assembly, as was demonstrated by non-denaturing PAGE. After use of mixtures of purified AO from H. polymorpha and P. pastoris active hybrid AO oligomers were formed. When prior to dissociation FAD was chemically removed from AO, reactivation or re-assembly did not occur independent of externally added FAD.

Substrate uptake and utilization by a marine ultramicrobacterium.

A facultatively oligotrophic ultramicrobacterium (strain RB2256) isolated from an Alaskan fjord by extinction dilution in seawater, was grown in batch culture and under single- and dual-substrate-limitation of alanine and glucose in a chemostat. The nature of the uptake systems, and the uptake kinetics and utilization patterns of alanine and glucose were investigated. Glucose uptake was inducible, the system exhibited a narrow substrate specificity, and part of the uptake system was osmotic-shock-sensitive. Half-saturation constants for glucose were between 7 and 74 microM during glucose limitation. The initial step in glucose metabolism was the synthesis of sugar polymers, even during glucose-limited growth. The alanine uptake system was constitutively expressed and was binding-protein-dependent. In addition to L-alanine, nine other amino acids inhibited accumulation of [14C]L-alanine, indicating broad substrate specificity of the alanine transporter. Half-saturation constants between 1.3 and 1.8 microM were determined for alanine uptake during alanine limitation. Simultaneous utilization of glucose and alanine occurred during substrate-limited growth in the chemostat, and during growth in batch culture at relatively high (mM) substrate concentrations. However, the half-saturation constant for alanine transport during dual-substrate-limitation, i.e. in the presence of glucose, increased almost fivefold. We conclude that mixed substrate utilization is an inherent property of this organism.

The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal.

Here we describe the identification of the targeting sequence of peroxisomal amine oxidase (AMO) of H. polymorpha. Deletion analysis revealed that essential targeting information is located within the extreme N-terminal 16 amino acids. Moreover, this sequence can direct a reporter protein to the peroxisomal matrix of H. polymorpha. The N-terminal 16 amino acids of AMO contain a sequence with strong homology to the conserved PTS2 sequence. Therefore, AMO is considered to be a PTS2 protein.

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

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.

The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins).

Two main types of peroxisomal targeting signals have been identified that reside either at the extreme C terminus (PTS1) or the N terminus (PTS2) of the protein. In the methylotrophic yeast Hansenula polymorpha the majority of peroxisomal matrix proteins are of the PTS1 type. Thus far, for H. polymorpha only amine oxidase (AMO) has been shown to contain a PTS2 type signal. In the present study we expressed H. polymorpha AMO under control of the strong endogenous alcohol oxidase promoter. Partial import of AMO into peroxisomes was observed in cells grown in methanol/(NH4)2SO4-containing medium. However, complete import of AMO occurred if the cells were grown under conditions that induce expression of the endogenous AMO gene. Similar results were obtained when the heterologous PTS2 proteins, glyoxysomal malate dehydrogenase from watermelon and thiolase from Saccharomyces cerevisiae, were synthesized in H. polymorpha. The import of PTS1 proteins, however, was not affected by the growth conditions. These results indicate that the reduced rate of AMO import in (NH4)2SO4-grown cells is not due to competition with PTS1 proteins for the same import pathway. Apparently, AMO is imported via a separate pathway that is induced by amines and functions for PTS2 proteins in general.

The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals.

We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PER1p contains both a carboxy- (PTS1) and an amino-terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle. In wild-type H. polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support.

Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide.

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.