Proteasome-dependent protein quality control of the peroxisomal membrane protein Pxa1p.

Peroxisomes are eukaryotic organelles that function in numerous metabolic pathways and defects in peroxisome function can cause serious developmental brain disorders such as adrenoleukodystrophy (ALD). Peroxisomal membrane proteins (PMPs) play a crucial role in regulating peroxisome function. Therefore, PMP homeostasis is vital for peroxisome function. Recently, we established that certain PMPs are degraded by the Ubiquitin Proteasome System yet little is known about how faulty/non-functional PMPs undergo quality control. Here we have investigated the degradation of Pxa1p, a fatty acid transporter in the yeast Saccharomyces cerevisiae. Pxa1p is a homologue of the human protein ALDP and mutations in ALDP result in the severe disorder ALD. By introducing two corresponding ALDP mutations into Pxa1p (Pxa1MUT), fused to mGFP, we show that Pxa1MUT-mGFP is rapidly degraded from peroxisomes in a proteasome-dependent manner, while wild type Pxa1-mGFP remains relatively stable. Furthermore, we identify a role for the ubiquitin ligase Ufd4p in Pxa1MUT-mGFP degradation. Finally, we establish that inhibiting Pxa1MUT-mGFP degradation results in a partial rescue of Pxa1p activity in cells. Together, our data demonstrate that faulty PMPs can undergo proteasome-dependent quality control. Furthermore, our observations may provide new insights into the role of ALDP degradation in ALD.

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.

Peroxisomal fatty acid beta-oxidation in relation to the accumulation of very long chain fatty acids in cultured skin fibroblasts from patients with Zellweger syndrome and other peroxisomal disorders.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD+. However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate. The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

Rhizomelic chondrodysplasia punctata. Deficiency of 3-oxoacyl-coenzyme A thiolase in peroxisomes and impaired processing of the enzyme.

The rhizomelic form of chondrodysplasia punctata (RCDP) is a peroxisomal disorder characterized biochemically by an impairment of plasmalogen biosynthesis and phytanate catabolism. We have now found that the maturation of peroxisomal 3-oxoacyl-CoA thiolase is impaired in fibroblasts from RCDP patients. To establish the subcellular localization of the 3-oxoacyl-CoA thiolase precursor protein, cultured skin fibroblasts were fractionated on a continuous Nycodenz gradient. Only a small amount of 3-oxoacyl-CoA thiolase activity was present in the catalase-containing (peroxisomal) fractions of RCDP fibroblasts in comparison with control fibroblasts. Moreover, the amount of thiolase protein in immunoblots of the catalase-containing fractions was below the limit of detection. Finally, the beta-oxidation of [14C]palmitoyl-CoA was found to be reduced in these fractions. We conclude that the mutation in RCDP leads to a partial deficiency of 3-oxoacyl-CoA thiolase activity in the peroxisomes and, concomitantly, an impairment in the ability to convert the precursor of this protein to the mature form. The reduction of 3-oxoacyl-CoA thiolase activity results in a decrease in the rate of peroxisomal beta-oxidation of palmitoyl-CoA. However, the capacity of the peroxisomes to oxidize very-long-chain fatty acids must be sufficient to prevent excessive accumulation of these compounds in vivo.

Identification of a peroxisomal ATP carrier required for medium-chain fatty acid beta-oxidation and normal peroxisome proliferation in Saccharomyces cerevisiae.

We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation in Saccharomyces cerevisiae. YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane. Disruption of the YPR128c gene results in impaired growth of the yeast with the medium-chain fatty acid (MCFA) laurate as a single carbon source, whereas normal growth was observed with the long-chain fatty acid (LCFA) oleate. MCFA but not LCFA beta-oxidation activity was markedly reduced in intact ypr128cDelta mutant cells compared to intact wild-type cells, but comparable activities were found in the corresponding lysates. These results imply that a transport step specific for MCFA beta-oxidation is impaired in ypr128cDelta cells. Since MCFA beta-oxidation in peroxisomes requires both ATP and CoASH for activation of the MCFAs into their corresponding coenzyme A esters, we studied whether YPR128cp is an ATP carrier. For this purpose we have used firefly luciferase targeted to peroxisomes to measure ATP consumption inside peroxisomes. We show that peroxisomal luciferase activity was strongly reduced in intact ypr128cDelta mutant cells compared to wild-type cells but comparable in lysates of both cell strains. We conclude that YPR128cp most likely mediates the transport of ATP across the peroxisomal membrane.

Studies on phytanic acid alpha-oxidation in rat liver and cultured human skin fibroblasts.

We have studied the alpha-oxidation of phytanic acid in rat liver and human skin fibroblasts in order to try to resolve the controversial issue of the subcellular site of alpha-oxidation of phytanic acid. The results show that isolated mitochondria are able to alpha-oxidize phytanic acid whereas isolated peroxisomes show no phytanic acid alpha-oxidation activity. Intact hepatocytes were found to alpha-oxidize phytanic acid at a rate which is more than 20-fold higher than the activity found in postnuclear supernatant fractions incubated under optimal conditions. The alpha-oxidation of phytanic acid was found to be sensitive to inhibitors of the respiratory chain and an uncoupler of oxidative phosphorylation. Furthermore, the alpha-oxidation of phytanic acid was found to be deficient in cultured human skin fibroblasts with an inherited deficiency of cytochrome c oxidase and in fibroblasts with a deficiency of functional peroxisomes. We conclude that mitochondria are indispensable for phytanic acid alpha-oxidation. Furthermore, we propose that one (or more) of the partial reactions in phytanic acid alpha-oxidation proceeds in peroxisomes leading to the concept that phytanic acid oxidation in the intact cell requires the participation of both mitochondria and peroxisomes.

Peroxisomal enzyme activities in the human hepatoblastoma cell line HepG2 as compared to human liver.

In order to develop an in vitro model allowing investigation of the long-term effects of hormones and other agents on peroxisomes in liver cells, we measured the activity of a series of peroxisomal enzyme activities in HepG2 cells, a proliferating cell line derived from a human hepatoblastoma. The results obtained show that although in absolute terms peroxisomal enzyme activities are lower in HepG2 cells as compared to human liver, relative activities were comparable in HepG2 and human liver, respectively. Furthermore, it is shown that peroxisomes can easily be isolated from HepG2 cells using density gradient centrifugation. It is concluded that HepG2 cells represent a good model system to study the characteristic (long-term) regulation and control of metabolism of human liver peroxisomes.

Localization of peroxisomal 3-oxoacyl-CoA thiolase in particles of varied density in rat liver: implications for peroxisome biogenesis.

In this paper we report on the subcellular localization of peroxisomal thiolase in rat liver using density-gradient centrifugation and immunoelectron microscopy. The results obtained show that peroxisomes display great biochemical heterogeneity and can not be regarded as one homogeneous population of particles. We conclude that rat liver contains at least three distinct populations of peroxisomes, which are present both in normal-fed rats as well in rats treated with a plasticizer, di-(2-ethylhexyl)phthalate, known to induce peroxisomes. The following types of peroxisomes could be discerned: (1) Low-density peroxisomal particles containing 69-kDa peroxisomal membrane protein (PMP), dihydroxyacetonephosphate acyltransferase (DHAPAT) and the precursor form of peroxisomal thiolase (44-kDa). (2) Intermediate-density peroxisomal particles containing 69-kDa peroxisomal membrane protein, dihydroxyacetonephosphate acyltransferase, both 41-kDa (mature) and 44-kDa (immature) peroxisomal thiolase, catalase and D-aminoacid oxidase. (3) High-density peroxisomes containing 69-kDa peroxisomal membrane protein, dihydroxyacetonephosphate acyltransferase, 41-kDa thiolase, catalase and D-aminoacid oxidase.

Subcellular localization of squalene synthase in human hepatoma cell line Hep G2.

Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.

Characteristics and subcellular localization of pristanoyl-CoA synthetase in rat liver.

We have investigated the activation of pristanic acid to its CoA-ester in rat liver. The results show that peroxisomes, mitochondria as well as microsomes contain pristanoyl-CoA synthetase activity. On the basis of competition experiments and immunoprecipitation studies using antibodies raised against rat liver microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3) we conclude that pristanic acid is activated by the same enzyme which activates long-chain fatty acids, i.e., long-chain fatty acyl-CoA synthetase.

2-Hydroxyphytanic acid oxidase activity in rat and human liver and its deficiency in the Zellweger syndrome.

Phytanic acid is a saturated, branched-chain fatty acid which as a consequence of the presence of a methyl group at the 3-position cannot be degraded by beta-oxidation. Instead, phytanic acid first undergoes alpha-oxidation to yield pristanic acid which can be degraded by beta-oxidation. The structure of the alpha-oxidation pathway and its subcellular localization has remained an enigma although there is convincing evidence that 2-hydroxyphytanic acid is an obligatory intermediate. We have now studied the degradation of 2-hydroxyphytanic acid in both rat and human liver. The results show that 2-hydroxyphytanic acid is converted to 2-ketophytanic acid in homogenates of rat as well as human liver. Detailed studies in rat liver showed that the enzyme involved is localized in peroxisomes accepting molecular oxygen as second substrate and producing H2O2. 2-Ketophytanic acid formation from 2-hydroxyphytanic acid was found to be strongly deficient in liver samples from Zellweger patients which lack morphologically distinguishable peroxisomes. The latter results not only provide an explanation for the elevated levels of 2-hydroxyphytanic acid in Zellweger patients but also suggest that the subcellular localization of 2-hydroxyphytanic acid dehydrogenation is identical in rat and man, i.e., in peroxisomes.

Catalase in cultured skin fibroblasts from patients with the cerebro-hepato-renal (Zellweger) syndrome: normal maturation in peroxisome-deficient cells.

We have compared the properties of catalase in cultured skin fibroblasts from patients with the cerebro-hepato-renal (Zellweger) syndrome, in which peroxisomes are deficient, with those of catalase in fibroblasts from control subjects. The enzymes from the two types of fibroblasts are indistinguishable with respect to kinetic properties, subunit size and molecular mass of the native enzyme. The turnover of the enzyme, measured by following the rate of reappearance of catalase activity in fibroblasts after irreversible inactivation of existing molecules by 3-aminotriazole treatment of the cells, was the same in Zellweger fibroblasts as in control cells. These findings indicate that normal maturation of catalase can occur in the soluble cytoplasm and provide an explanation for the occurrence of extra-peroxisomal catalase in tissues and cells.

Studies on the peroxisomal oxidation of palmitate and lignocerate in rat liver.

We have investigated the pathways involved in the peroxisomal oxidation of palmitate and lignocerate, measured as the cyanide-insensitive formation of acetyl units, in rat-liver homogenates. The peroxisomal beta-oxidation of both fatty acids is dependent on the presence of ATP, coenzyme A, NAD+ and Mg2+. However, there is a striking difference in the dependence of the rate of oxidation of the two substrates on the concentration of the individual cofactors, especially ATP. The peroxisomal beta-oxidation of lignocerate was inhibited to a progressively greater extent by increasing concentrations of palmitate and vice versa. Activation of lignoceric acid to lignoceroyl-CoA, however, was not inhibited by increasing concentrations of palmitate, and vice versa. It can be concluded that the peroxisomal palmitate and lignocerate beta-oxidation pathways differ in at least one enzymic reaction (the synthetase), but that the two pathways share at least one common step.

Subcellular fractionation of cultured normal human melanocytes: new insights into the relationship of melanosomes with lysosomes and peroxisomes.

In order to obtain information on the disputed nature of melanosomes a comparison was made between the localization of melanosomal markers with those of other well-defined subcellular organelles such as lysosomes and peroxisomes. The distribution of marker enzymes was studied using two different density gradient systems, i.e., Percoll and Nycodenz. Furthermore, the subcellular localization of various types of antigens was analyzed using indirect immunofluorescence and immuno-electron microscopy. All methods revealed the existence of partial co-localization of melanosomal and lysosomal proteins and different localization of peroxisomal markers. The results suggest that melanosomes may share a common origin with lysosomal structures.

Mechanism of the stimulation of respiration by fatty acids in rat liver.

The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 micrograms/ml). With carboxyatractyloside (100 microM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50-70 and 50-60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50-60%) of this ATP is utilized by the (Na+ + K+)-ATPase.

Metabolic aspects of peroxisomal disorders.

In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in one or more peroxisomal functions. This paper discusses the current state of knowledge on these disorders with particular emphasis on the metabolic abnormalities in these diseases.

Prenatal diagnosis of Zellweger syndrome by measurement of very long chain fatty acid (C26:0) beta-oxidation in cultured chorionic villous fibroblasts: implications for early diagnosis of other peroxisomal disorders.

In this paper we show that cultured chorionic villous fibroblasts efficiently catalyse the peroxisomal beta-oxidation of hexacosanoic acid (cerotic acid), a saturated very long chain fatty acid containing 26 carbon atoms. Hexacosanoic beta-oxidation was found to be strongly impaired in cultured chorionic villous fibroblasts from a Zellweger foetus. This finding indicates that measurement of peroxisomal beta-oxidation can be used (in addition to measurement of acyl-CoA:dihydroxyacetone phosphate acyltransferase, de novo plasmalogen biosynthesis, the amount of particle-bound catalase and phytanic acid oxidase) for prenatal diagnosis in the first trimester of Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy. The method should be equally applicable to the early prenatal diagnosis of disorders in which there is a deficiency of a single peroxisomal beta-oxidation enzyme. Such diseases include X-linked adrenoleukodystrophy (peroxisomal very long chain fatty acyl CoA ligase deficiency), 'pseudo-Zellweger syndrome' (peroxisomal 3-oxoacyl-CoA thiolase deficiency) and 'pseudo-neonatal adrenoleukodystrophy' (acyl-CoA oxidase deficiency).

Human liver L-alanine-glyoxylate aminotransferase: characteristics and activity in controls and hyperoxaluria type I patients using a simple spectrophotometric method.

We have studied the characteristics of human liver alanine-glyoxylate aminotransferase, which is deficient in hyperoxaluria type I, an inherited disorder of glyoxylate metabolism. The enzyme was optimally active at pH 8.0 showing apparent Km values for L-alanine and glyoxylate of 8.3 and 1.3 mmol/l, respectively. Activity was found to proceed linearly for up to 4 h. Measurements under these optimal conditions enabled the biochemical diagnosis of hyperoxaluria type I to be made via enzyme activity measurements in percutaneous needle biopsy specimens of liver tissue.

Accumulation and impaired in vivo metabolism of di- and trihydroxycholestanoic acid in two patients.

Two patients with a suspected peroxisomal disorder on the basis of neurological, craniofacial, hepatological and other abnormalities were studied. The phenotype of both girls was remarkably similar from birth until age 1.5 yr. Detailed studies in plasma revealed normal plasma very-long-chain fatty acids but the presence of di- and trihydroxycholestanoic acids and the C29-dicarboxylic bile acid, all known to occur in plasma from Zellweger patients. These results suggest an isolated defect in the peroxisomal beta-oxidation of the side chains of the cholestanoic acids. Activation of trihydroxycholestanoic acid and beta-oxidation of trihydroxycholestanoyl-CoA, measured in a liver biopsy, were normal, however, as was the peroxisomal beta-oxidation of palmitate. Although the molecular defect remains unknown, the results stress the importance of performing multiple analyses in any patient suspected to suffer from a peroxisomal disorder and indicate that screening for peroxisomal disorders based upon analysis of only plasma very long chain fatty acids with or without analysis of erythrocyte plasmalogen levels, may be inadequate.

Peroxisomal very long-chain fatty acid beta-oxidation in human skin fibroblasts: activity in Zellweger syndrome and other peroxisomal disorders.

Since very long-chain fatty acids with a chain length of 24 carbons or more are known to accumulate in tissues and body fluids from patients with the cerebrohepato-renal (Zellweger) syndrome, infantile Refsum disease, neonatal adrenoleukodystrophy and X-linked adrenoleukodystrophy, we studied very long-chain fatty acid oxidation in cultured skin fibroblasts from these patients. In this paper, we report that in accordance with earlier results the first step in the beta-oxidation of the very long-chain fatty acid lignoceric acid (C24:0) primarily occurs in peroxisomes in control human skin fibroblasts. Furthermore, it was found that peroxisomal lignoceric acid beta-oxidation was strongly deficient in fibroblasts from patients with Zellweger syndrome, infantile Refsum disease, neonatal and X-linked adrenoleukodystrophy, which explains for the accumulation of very long-chain fatty acids in all four disease entities. In Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy the impairment in peroxisomal very long-chain fatty acid beta-oxidation is probably caused by a strong deficiency of all peroxisomal beta-oxidation enzyme proteins due to a deficiency of peroxisomes.