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

Reinvestigation of peroxisomal 3-ketoacyl-CoA thiolase deficiency: identification of the true defect at the level of d-bifunctional protein

In this report, we reinvestigate the only patient ever reported with a deficiency of peroxisomal 3-ketoacyl-CoA thiolase (THIO). At the time when they were described, the abnormalities in this patient, which included accumulation of very-long-chain fatty acids and the bile-acid intermediate trihydroxycholestanoic acid, were believed to be the logical consequence of a deficiency of the peroxisomal β-oxidation enzyme THIO. In light of the current knowledge of the peroxisomal β-oxidation system, however, the reported biochemical aberrations can no longer be explained by a deficiency of this thiolase. In this study, we show that the true defect in this patient is at the level of d-bifunctional protein (DBP). Immunoblot analysis revealed the absence of DBP in postmortem brain of the patient, whereas THIO was normally present. In addition, we found that the patient had a homozygous deletion of part of exon 3 and intron 3 of the DBP gene, resulting in skipping of exon 3 at the cDNA level. Our findings imply that the group of single–peroxisomal β-oxidation–enzyme deficiencies is limited to straight-chain acyl-CoA oxidase, DBP, and α-methylacyl-CoA racemase deficiency and that there is no longer evidence for the existence of THIO deficiency as a distinct clinical entity

Peroxisomal NAD(H) homeostasis in the yeast debaryomyces hansenii depends on two redox shuttles and the NAD+ carrier, Pmp47

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the β-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD+ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during β-oxidation to the cytosol. Pmp47 is the first identified NAD+ carrier in yeast peroxisomes

Complementation analysis of fibroblasts from peroxisomal fatty acid oxidation deficient patients shows high frequency of bifunctional enzyme deficiency plus intragenic complenetation: unequivocal evidence for differential defects in the same enzyme proten

In the last few years many patients have been reported with a defect in peroxisomal fatty acid beta-oxidation of unknown origin. Using a combined approach based on direct activity measurements of straight-chain acyl-CoA oxidase and complementation analysis after somatic cell fusion of fibroblasts, we have now classified 13 patients into 4 distinct groups representing different gene defects. Remarkably, we found intragenic complementation in group 2 so that group 2 is in fact made up of 3 distinct subgroups. The underlying basis for this peculiar phenomenon probably has to do with the fact that bifunctional protein harbors two catalytic activities including enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. In group 2A enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase are defective whereas in group 2B and 2C either the hydratase or 3-hydroxyacyl-CoA dehydrogenase component of the bifunctional protein is deficien

Cytosolic aspartate aminotransferase encoded by the AAT2 gene is targeted to the peroxisomes in oleate-grown Saccharomyces cerevisiae

Fatty acid beta-oxidation in peroxisomes requires the continued uptake of fatty acids or their derivatives into peroxisomes and export of beta-oxidation products plus oxidation of NADH to NAD. In an earlier study we provided evidence for the existence of an NAD(H) redox shuttle in which peroxisomal malate dehydrogenase plays a pivotal role. In analogy to the NAD(H)-redox-shuttle systems in mitochondria we have investigated whether a malate/aspartate shuttle is operative in peroxisomes. The results described in this paper show that peroxisomes of oleate-grown Saccharomyces cerevisiae contain aspartate aminotransferase (AAT) activity. Whereas virtually all cellular AAT activity was peroxisomal in oleate-grown cells, we found that in glucose-grown cells most of the AAT activity resided in the cytosol. We demonstrate that the gene AAT2 codes for the cytosolic and peroxisomal AAT activities. Disruption of the AAT2 gene did not affect growth on oleate. Furthermore beta-oxidation of palmitate was normal. These results indicate that AAT2 is not essential for the peroxisomal NAD(H) redox shuttl

Peroxisomal fatty acid α- and β-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases

Peroxisomes are subcellular organelles with an indispensable role in cellular metabolism. The importance of peroxisomes for humans is stressed by the existence of a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. Most of these functions have to do with lipid metabolism including the alpha- and beta-oxidation of fatty acids. Here we describe the current state of knowledge about peroxisomal fatty acid alpha- and beta-oxidation with particular emphasis on the following: (1) the substrates beta-oxidized in peroxisomes; (2) the enzymology of the alpha- and beta-oxidation systems; (3) the permeability properties of the peroxisomal membrane and the role of the different transporters therein; (4) the interaction with other subcellular compartments, including the mitochondria, which are the ultimate site of NADH re-oxidation and full degradation of acetyl-CoA to CO(2) and water; and (5) the different disorders of peroxisomal alpha- and beta-oxidatio