Classical genetic manipulation.
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Animals↗
Biomedical subjects
H R Waterham
Publications and source records attributed to H R Waterham.
Peroxisomal targeting, import, and assembly of alcohol oxidase in Pichia pastoris.
Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX-HA). Coexpression of AOX-HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX-HA present in octameric active AOX, suggesting that newly synthesized AOX-HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.
Alcohol Oxidoreductases↗
Functional characterization of mitochondrial carnitine palmitoyltransferases I and II expressed in the yeast Pichia pastoris.
The rate-limiting step in beta oxidation is the conversion of long-chain acyl-CoA to acylcarnitine, a reaction catalyzed by the outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) and inhibited by malonyl-CoA. The acylcarnitine is then translocated across the inner mitochondrial membrane by the carnitine/acylcarnitine translocase and converted back to acyl-CoA by CPTII. Although CPTII has been examined in detail, studies on CPTI have been hampered by an inability to purify CPTI in an active form from CPTII. In particular, it has not been conclusively demonstrated that CPTI is even catalytically active, or whether sensitivity of CPTI to malonyl-CoA is an intrinsic property of the enzyme or is contained in a separate regulatory subunit that interacts with CPTI. To address these questions, the genes for CPTI and CPTII were separately expressed in Pichia pastoris, a yeast with no endogenous CPT activity. High levels of CPT activity were present in purified mitochondrial preparations from both CPTI- and CPTII-expressing strains. Furthermore, CPTI activity was highly sensitive to inhibition by malonyl-CoA while CPTII was not. Thus, CPT catalytic activity and malonyl-CoA sensitivity are contained within a single CPTI polypeptide in mammalian mitochondrial membranes. We describe the kinetic characteristics for the yeast-expressed CPTs, the first such report for a CPTI enzyme in the absence of CPTII. Yeast-expressed CPTI is inactivated by detergent solubilization. However, removal of the detergent in the presence of phospholipids resulted in the recovery of malonyl-CoA-sensitive CPTI activity, suggesting that CPTI requires a membranous environment. CPTI is thus reversibly inactivated by detergents.
Acyl Coenzyme A↗
Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter.
We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P(GAP)) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth. Expression of beta-lactamase under control of P(GAP) in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P(AOX1)) in methanol-grown cells. As an example of the use of P(GAP), we showed that beta-lactamase synthesized under transcriptional control of P(GAP) is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P(GAP) has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P(AOX1) in P. pastoris.
Alcohol Oxidoreductases↗
Peroxisome biogenesis.
Peroxisomes are eukaryotic organelles that are the subcellular location of important metabolic reactions. In humans, defects in the organelle's function are often lethal. Yet, relative to other organelles, little is known about how cells maintain and propagate peroxisomes or how they direct specific sets of newly synthesized proteins to these organelles (peroxisome biogenesis/assembly). In recent years, substantial progress has been made in elucidating aspects of peroxisome biogenesis and in identifying PEX genes whose products, peroxins, are essential for one or more of these processes. The most progress has been made in understanding the mechanism by which peroxisome matrix proteins are imported into the organelles. Signal sequences responsible for targeting proteins to the organelle have been defined. Potential signal receptor proteins, a receptor docking protein and other components of the import machinery have been identified, along with insights into how they operate. These studies indicate that multiple peroxisomal protein-import mechanisms exist and that these mechanisms are novel, not simply variations of those described for other organelles.
Biological Transport, Active↗
The Pichia pastoris PER6 gene encodes the yeast homologue of human PAF-1.
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Fungal Proteins↗
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.
Amino Acid Sequence↗
The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.
We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
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.
Alcohol Oxidoreductases↗
The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation.
We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Peroxisome biogenesis in the yeast Hansenula polymorpha is controlled by a complex set of interacting gene products.
We have studied the genetic interactions between mutant alleles in 12 genes, designated PER1-PER12, which are essential for peroxisome biogenesis in the yeast Hansenula polymorpha. Recessive mutations in any of these genes determined three different morphological phenotypes: (i) complete absence of peroxisomes (Per-); (ii) presence of small peroxisomes in conjunction with a major fraction of peroxisomal matrix proteins in the cytosol (Pim-); and (iii) presence of peroxisomes with aberrant crystalline matrix substructure (Pss-). Extensive complementation analysis showed many cases of noncomplementation--that is, diploids that contained both wild-type and mutant alleles of two different PER genes were unable to grow on methanol and showed peroxisomal defects. The observed cases of unlinked noncomplementation appeared to be gene and allele specific and were predominantly observed at lower temperatures (cold sensitive). The genetic results obtained were used to formulate a model of PER gene product interactions. In this model, five PER gene products are key or core components of the complex. Other PER gene products appear to play a more peripheral role.
Alleles↗
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.
Blotting, Western↗
Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.
We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.
Blotting, Western↗
Peroxisome biogenesis in Hansenula polymorpha: different mutations in genes, essential for peroxisome biogenesis, cause different peroxisomal mutant phenotypes.
In Hansenula polymorpha, different monogenic recessive mutations mapped in either of two previously identified genes, PER1 and PER3, produced different peroxisomal mutant phenotypes. Among five per1 mutants, four showed a Pim- phenotype: the cells contained few small peroxisomes while the bulk of the matrix enzymes resided in the cytosol. One of these mutants, per1-124 had an enhanced rate of peroxisome proliferation. The fifth mutant completely lacked peroxisomes (Per- phenotype). Of seven per3 mutants, four displayed a Pim- phenotype, two others a Per- phenotype, while one mutant showed pH-dependent growth on methanol and was affected in oligomerization of peroxisomal matrix protein. Thus, the protein products of both PER1 and PER3 genes appear to be essential in different aspects of peroxisome assembly/proliferation.
Genes, Fungal↗
Development of multipurpose peroxisomes in Candida boidinii grown in oleic acid-methanol limited continuous cultures.
We have studied the development and metabolic significance of peroxisomes in the yeast Candida boidinii following adaptation of the organism to cultivation conditions which require the simultaneous presence and activity of two independent peroxisome-mediated pathways for growth. After the addition of methanol to oleic acid-grown cells at late exponentional growth, a number of new small peroxisomes developed which, apart from the presence of beta-oxidation enzymes, were characterized by the presence of enzymes involved in methanol metabolism (alcohol oxidase and dihydroxyacetone synthase). The latter proteins, however, were absent in the larger organelles which were originally present in the oleic acid-grown cells prior to the addition of methanol and which contained only enzymes of the beta-oxidation pathway. Subsequent experiments on cells from continuous cultures grown on a mixture of oleic acid and methanol at steady-state conditions revealed that both the enzymes of the beta-oxidation pathway and those involved in methanol metabolism were found in one and the same compartment. Thus, under these conditions the cells contained peroxisomes which were concurrently involved in the metabolism of two different carbon sources simultaneously used for growth. Our results indicated that the heterogeneity in the peroxisomal population of a single cell, observed in the transient state following the addition of methanol, is only temporary and due to heterogeneity among these organelles with respect to their capacity to incorporate newly synthesized matrix proteins.
Acetyl-CoA C-Acetyltransferase↗
Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts.
The possible acidic nature of the peroxisomal matrix present in intact yeast cells was studied immunocytochemically, using the weak base DAMP as a probe. Spheroplasts of methanol-grown Candida boidinii and Hansenula polymorpha were regenerated and incubated with DAMP. After immunogold labelling, using antibodies against DAMP, a specific accumulation of gold particles was observed on the peroxisomal profiles. This labelling was absent in controls, performed in the presence of ionophores or chloroquine. These results support earlier observations, that in intact cells a pH-gradient exists across the peroxisomal membrane. Experiments, carried out on osmotically swollen spheroplasts indicated that maintenance of this pH-gradient is strongly related to the cell's integrity.
Candida↗