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.

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.

Yeast peroxisomes: function and biogenesis of a versatile cell organelle.

Yeast peroxisomes harbour enzymes involved in the metabolism of specific growth substrates. Sequestration of these enzymes increases the efficiency of such pathways. Currently, 16 genes involved in peroxisome biogenesis have been identified, and analysis of their products suggests novel mechanisms for organelle assembly and protein translocation.

Deflavination of flavo-oxidases by nucleophilic reagents.

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.

Electron tomography of mitochondria after the arrest of protein import associated with Tom19 depletion.

In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA). Using intermediate-voltage electron tomography, we have generated three-dimensional reconstructions of 28 FPA-treated mitochondria at four time points (0-32 h) after the addition of FPA. We determined that the cristae surface area and volume were lost in a roughly linear manner. A decrease in mitochondrial volume was not observed until after 16 h of FPA treatment. The inner boundary membrane did not appear to shrink or contract away from the outer membrane. Interestingly, the close apposition of these membranes remained over the entire periphery, even after all of the cristae had disappeared. The different dynamics of the shrinkage of cristae membrane and inner boundary membrane has implications for compartmentalization of electron transport proteins. Two structurally distinct types of contact sites were observed, consistent with recently published work. We determined that the cristae in the untreated (control) mitochondria are all lamellar. The cristae of FPA-treated mitochondria retain the lamellar morphology as they reduce in size and do not adopt tubular shapes. Importantly, the crista junctions exhibit tubular as well as slot-like connections to the inner boundary membrane, persisting until the cristae disappear, indicating that their stability is not dependent on continuous protein import through the complex containing Tom19.

Selective peroxisome degradation in Yarrowia lipolytica after a shift of cells from acetate/oleate/ethylamine into glucose/ammonium sulfate-containing media.

We have shown that peroxisomes of the yeast Yarrowia lipolytica are subject to specific degradation after exposure of acetate/oleate-grown cells to glucose excess conditions. Electron microscopic analysis has revealed that the peroxisomes were degraded by uptake in the vacuole. Our results suggest that peroxisomes are taken up by macroautophagic processes, because sequestration of individual peroxisomes, which occurs typically at the beginning of microautophagy, was never observed. The observation that a peroxisomal amine oxidase activity is specifically induced by ethylamine was used for the development of a plate assay screening procedure to isolate peroxisome degradation-defective mutants.

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.

Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole.

Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumulation of ubiquitin-protein conjugates in this cellular organelle.

Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase.

Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods. The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 A. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.

Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha.

We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector downstream of the inducible H. polymorpha alcohol oxidase promoter. During methylotrophic growth, pre-gMDH was synthesized and imported into peroxisomes, where it was enzymatically active. The apparent molecular mass of the protein located in H. polymorpha peroxisomes was equal to that of pre-gMDH (41 kDa), indicating that N-terminal processing of the transit peptide had not occurred in the yeast.

Peroxisomal remnants in peroxisome-deficient mutants of the yeast Hansenula polymorpha [corrected].

We have analyzed the presence of peroxisomal remnants ('ghosts') in three peroxisome-deficient (per) mutants of the yeast Hansenula polymorpha, namely delta per4, delta per5 and delta per10. Under peroxisome-inducing growth conditions peroxisomal membrane proteins (PMPs) were normally synthesized in cells of these mutants. In addition, these cells contained clusters of small membraneous vesicles, which were absent in cells grown under peroxisome-repressing growth conditions. These structures displayed typical peroxisomal properties in that they proliferated upon overproduction of Per8p, the H. polymorpha peroxisome proliferation factor. Moreover, in delta per4 and delta per5 these vesicles were susceptible to glucose-induced proteolytic degradation.

Peroxisome biogenesis and degradation in yeast: a structure/function analysis.

In yeast, peroxisomes are the site of specific catabolic pathways that characteristically include hydrogen peroxide producing oxidases and catalase. During the last 10 years, much progress has been made in unravelling the molecular mechanisms involved in the biogenesis of this organelle. At present, 23 different genes (PEX genes) have been identified that are involved in different aspects of peroxisome biogenesis (e.g., proliferation, formation of the peroxisomal membrane, import of matrix proteins). The principles of peroxisome degradation are still much less understood. Recently, the first yeast mutants affected in this process have become available and used to clone corresponding genes by functional complementation. In this paper, an overview is presented of the research on yeast peroxisomes, focusing on recent achievements in the molecular aspects of peroxisome development, function, and turnover.

A morphological view on mitochondrial protein targeting.

Mitochondrial protein targeting includes both intramitochondrial sorting of proteins encoded by the organellar genome and import and subsequent sorting of nuclear encoded precursor proteins. Only a few proteins are encoded by the mitochondrial genome and synthesized in the organellar matrix. These include predominantly inner membrane proteins that are perhaps co-translationally inserted into this membrane. Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane. Ultrastructurally, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established. The majority of the mitochondrial proteins are nuclear encoded and synthesized as precursors in the cytosol. Electron microscopic studies revealed that import of precursor proteins is generally confined to sites where both mitochondrial envelope membranes are closely apposed. In line with these observations, biochemical studies indicated that precursor proteins destined for the inner membrane or matrix have to interact with the energized inner membrane to allow complete passage of the precursor through the outer membrane. As a consequence, the mitochondrial envelope membranes have to be in close proximity at protein import sites. In isolated mitochondria distinct sites (designated as contact sites) exist where both envelope membranes are closely apposed and presumably stably associated. In situ, however, mitochondrial boundary membranes are in close proximity over large areas that cover almost the entire mitochondrial periphery. Consequently, the relative area of the mitochondrial surface, where both boundary membranes are in sufficient proximity for allowing protein translocation, is generally larger in situ compared to that in isolated organelles. Immunocytochemical localization studies showed a rather random distribution of components of the mitochondrial protein translocation machinery over the entire mitochondrial surface and not confined to contact sites. Based on these ultrastructural data and recent biochemical findings we propose that mitochondrial protein import sites are dynamic in nature and include relatively labile regions of close association of the boundary membranes. In vitro, however, mitochondrial protein import may preferentially take place at or near the presumably stable contact sites.

Hansenula polymorpha: an attractive model organism for molecular studies of peroxisome biogenesis and function.

In wild-type Hansenula polymorpha the proliferation of peroxisomes in induced by various unconventional carbon- and nitrogen sources. Highest induction levels, up to 80% of the cytoplasmic volume, are observed in cells grown in methanol-limited chemostat cultures. Based on our accumulated experience, we are now able to precisely adjust both the level of the peroxisome induction as well as their protein composition by specific adaptations in growth conditions. During the last few years a series of "peroxisome-deficient (per) mutants of H. polymorpha have been isolated and characterized. Phenotypically these mutants are characterized by the fact that they are not able to grow on methanol. Three mutant phenotypes were defined on the basis of morphological criteria, namely: (a) mutants completely lacking peroxisomes (Per-;13 complementation groups); (b) mutants containing few small peroxisomes which are partly impaired in the peroxisomal import of matrix proteins (Pim-; five complementation groups); and (c) mutants with aberrations in the peroxisomal substructure (Pss-; two complementation groups). In addition, several conditional Per-, Pim- and Pss- mutants have been obtained. In all cases the mutant phenotype was shown to be caused by a recessive mutation in one gene. However, we observed that different mutations in one gene may cause different morphological mutant phenotypes. A detailed genetic analysis revealed that several PER genes, essential for peroxisome biogenesis, are tightly linked and organized in a hierarchical fashion. The use of both constitual and conditional per mutants in current and future studies of the molecular mechanisms controlling peroxisome biogenesis and function is discussed.

Selective inactivation of alcohol oxidase in two peroxisome-deficient mutants of the yeast Hansenula polymorpha.

We have studied selective inactivation of alcohol oxidase (AO) in two peroxisome-deficient (PER) mutants of the yeast Hansenula polymorpha. In these mutants high activities of cytosolic AO are induced by different growth conditions. At enhanced expression rates AO is arranged in large crystalloids in the cytosol, whereas smaller crystalloids are often observed inside the nucleus. Transfer of cells of the PER mutant 125-2E, which completely lacks peroxisomes, to glucose-excess conditions did not lead to degradative inactivation of AO and catalase as observed in wild-type (WT) cells used as a control. The gradual decrease in enzyme activities in the PER mutant could be accounted for by dilution of existing enzyme into newly formed cells as a result of growth. Morphologically, degradation of the cytosolic crystalloids was also not observed. Similar results were obtained with a second PER mutant (strain 124-2D), impaired in the import of peroxisomal matrix proteins. This mutant is characterized by the presence of small peroxisomes and large cytosolic AO crystalloids. Upon a shift of cells to glucose-excess conditions only part of the small peroxisomes present in these cells were degraded by mechanisms similar to those observed in WT cells placed under identical conditions. These results indicate that degradative inactivation of AO in H. polymorpha is strictly dependent on the localization of the enzyme inside peroxisomes and furthermore suggests that the mechanisms triggering this process are not directed against AO protein, but instead, to the membrane surrounding the organelle. Transfer of cells to methanol- or ethanol-containing media both resulted in modification inactivation of AO.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review.

Alcohol oxidase (AO) catalyses the first step of methanol metabolism in yeasts. In vivo the enzyme is compartmentalized in special cell compartments, called peroxisomes. The enzyme along with the organelles are induced during growth of methylotrophic yeasts on methanol as the sole carbon source. Like all other peroxisomal matrix proteins, AO is encoded by a nuclear gene. Expression of the protein is regulated by a repression/derepression mechanism, but also by induction. Inactive monomeric precursor protein is synthesized in the cytosol and subsequently imported post-translationally into peroxisomes without further processing. Assembly into the active homo-octameric enzyme and binding of the prosthetic group flavin adenine dinucleotide occurs inside the organelle. When enhanced concentration of octameric alcohol oxidase are present in the organelles, the enzyme may form a crystalloid. Oligomerization is not dependent on translocation of AO precursors into their target organelle since octameric, active AO is detected in the cytosol and nucleus of peroxisome-deficient mutants of Hansenula polymorpha: at high expression rates large cytosolic AO crystalloids are formed, which occasionally are also encountered inside the nucleus of such mutants. This paper summarizes recent findings and views on the mechanisms involved in synthesis, import, assembly and crystallization of this important peroxisomal enzyme.

Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study.

We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (SR = 12.5 mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanol-metabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H2O2-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.