Marlene van den Berg

Saccharomyces cerevisiae Pex3p and Pex19p are required for proper localization and stability of peroxisomal membrane proteins

The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Δ and pex19Δ, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexΔ mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER‐accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.

Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p.

In Saccharomyces cerevisiae, β‐oxidation of fatty acids is confined to peroxisomes. The acetyl‐CoA produced has to be transported from the peroxisomes via the cytoplasm to the mitochondrial matrix in order to be degraded to CO2 and H2O. Two pathways for the transport of acetyl‐CoA to the mitochondria have been proposed. The first involves peroxisomal conversion of acetyl‐CoA into glyoxylate cycle intermediates followed by transport of these intermediates to the mitochondria. The second pathway involves peroxisomal conversion of acetyl‐CoA into acetylcarnitine, which is subsequently transported to the mitochondria. Using a selective screen, we have isolated several mutants that are specifically affected in the second pathway, the carnitine‐dependent acetyl‐CoA transport from the peroxisomes to the mitochondria, and assigned these CDAT mutants to three different complementation groups. The corresponding genes were identified using functional complementation of the mutants with a genomic DNA library. In addition to the previously reported carnitine acetyl‐CoA transferase (CAT2), we identified the genes for the yeast orthologue of the human mitochondrial carnitine acylcarnitine translocase (YOR100C or CAC) and for a transport protein (AGP2) required for carnitine transport across the plasma membrane.

A Conserved Cysteine Is Essential for Pex4p-dependent Ubiquitination of the Peroxisomal Import Receptor Pex5p

The peroxisomal protein import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and -independent manner. Here we show that the two types of ubiquitination target different residues in the NH2-terminal region of Pex5p and we identify Pex4p (Ubc10p) as the ubiquitin-conjugating enzyme required for Ubc4p-independent ubiquitination. Whereas Ubc4p-dependent ubiquitination occurs on two lysine residues, Pex4p-dependent ubiquitination neither requires lysine residues nor the NH2-terminal α-NH2 group. Instead, a conserved cysteine residue appears to be essential for both the Pex4p-dependent ubiquitination and the overall function of Pex5p. In addition, we show that this form of ubiquitinated Pex5p is susceptible to the reducing agent β-mercaptoethanol, a compound that is unable to break ubiquitin-NH2 group linkages. Together, our results strongly suggest that Pex4p-dependent ubiquitination of Pex5p occurs on a cysteine residue.

Endoplasmic Reticulum-directed Pex3p Routes to Peroxisomes and Restores Peroxisome Formation in a Saccharomyces cerevisiae pex3Δ Strain

Recent studies on the sorting of peroxisomal membrane proteins challenge the long-standing model in which peroxisomes are considered to be autonomous organelles that multiply by growth and division. Here, we present data lending support to the idea that the endoplasmic reticulum (ER) is involved in sorting of the peroxisomal membrane protein Pex3p, a protein required early in peroxisome biogenesis. First, we show that the introduction of an artificial glycosylation site into the N terminus of Pex3p leads to partial N-linked core glycosylation, indicative of insertion into the ER membrane. Second, when FLAG-tagged Pex3p is equipped with an ER targeting signal, it can restore peroxisome formation in pex3Δ cells. Importantly, FLAG antibodies that specifically recognize the processed Pex3p show that the signal peptide of the fusion protein is efficiently cleaved off and that the processed protein localizes to peroxisomes. In contrast, a Pex3p construct in which cleavage of the signal peptide is blocked by a mutation localizes to the ER and the cytosol and cannot complement pex3Δ cells. Together, these results strongly suggest that ER-targeted Pex3p indeed routes via the ER to peroxisomes, and we hypothesize that this pathway is also used by endogenous Pex3p.

ER-directed Pex3p routes to peroxisomes and restores peroxisome formation in a Saccharomyces cerevisiae pex3ù strain

Recent studies on the sorting of peroxisomal membrane proteins challenge the long-standing model in which peroxisomes are considered to be autonomous organelles that multiply by growth and division. Here, we present data lending support to the idea that the endoplasmic reticulum (ER) is involved in sorting of the peroxisomal membrane protein Pex3p, a protein required early in peroxisome biogenesis. First, we show that the introduction of an artificial glycosylation site into the N terminus of Pex3p leads to partial N-linked core glycosylation, indicative of insertion into the ER membrane. Second, when FLAG-tagged Pex3p is equipped with an ER targeting signal, it can restore peroxisome formation in pex3Δ cells. Importantly, FLAG antibodies that specifically recognize the processed Pex3p show that the signal peptide of the fusion protein is efficiently cleaved off and that the processed protein localizes to peroxisomes. In contrast, a Pex3p construct in which cleavage of the signal peptide is blocked by a mutation localizes to the ER and the cytosol and cannot complement pex3Δ cells. Together, these results strongly suggest that ER-targeted Pex3p indeed routes via the ER to peroxisomes, and we hypothesize that this pathway is also used by endogenous Pex3p.

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

ABSTRACT We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation inSaccharomyces cerevisiae. YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane. Disruption of theYPR128c 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 β-oxidation activity was markedly reduced in intact ypr128cΔ 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 β-oxidation is impaired in ypr128cΔ cells. Since MCFA β-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 ypr128cΔ 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.

Pex10p functions as an E3 ligase for the Ubc4p-dependent ubiquitination of Pex5p.

The Saccharomyces cerevisiae (Sc) PTS1 import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and a Pex4p (Ubc10p)-dependent manner. Both of these modifications require the RING domain-containing protein Pex10p in vivo, but the actual role this protein plays in the ubiquitination of Pex5p has so far, remained enigmatic. Here, we report that the RING domain of Pex10p exhibits E(3) ligase activity in vitro, in combination with the human E(2) enzyme UbcH5a, a homologue of ScUbc4p, but not when ScPex4p was used as an E(2) enzyme in the reaction. We have further characterised Pex10ps E(3) ligase activity using mutants designed to disturb this activity and show that Pex10p acts as the E(3) ligase for Ubc4p-dependent ubiquitination of Pex5p but not Pex4p-dependent ubiquitination in vivo. These data imply that the two distinct Pex5p modifications require different E(3) ligases, as well as different E(2) enzymes.

Peroxisomal fatty acid beta-oxidation is not essential for virulence of Candida albicans.

ABSTRACT Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid β-oxidation pathway. Here, we asked whether fatty acid β-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Δ/pex5Δ mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Δ/fox2Δ mutant, which lacks the second enzyme of the β-oxidation pathway. Both mutant strains had strongly reduced β-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Δ/fox2Δ mutant, and not the pex5Δ/pex5Δ mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2Δ/fox2Δ mutant was comparable to that of the icl1Δ/icl1Δ mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid β-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2Δ/fox2Δ mutant is largely due to a dysfunctional glyoxylate cycle.

Insights into ubiquitin-conjugating enzyme/ co-activator interactions from the structure of the Pex4p:Pex22p complex

Ubiquitin‐conjugating enzymes (E2s) coordinate distinct types of ubiquitination via specific E3 ligases, to a large number of protein substrates. While many E2 enzymes need only the presence of an E3 ligase for substrate ubiquitination, a number of E2s require additional, non‐canonical binding partners to specify their function. Here, we have determined the crystal structure and function of an E2/co‐activator assembly, the Pex4p:Pex22p complex. The peroxisome‐associated E2 enzyme Pex4p binds the peroxisomal membrane protein Pex22p through a binding site that does not overlap with any other known interaction interface in E2 enzymes. Pex22p association enhances Pex4ps ability to transfer ubiquitin to a substrate in vitro, and Pex22p binding‐deficient forms of Pex4p are unable to ubiquitinate the peroxisomal import receptor Pex5p in vivo. Our data demonstrate that the Pex4p:Pex22p assembly, and not Pex4p alone, functions as the E2 enzyme required for Pex5p ubiquitination, establishing a novel mechanism of E2 enzyme regulation.

The activity of the glyoxylate cycle in peroxisomes of Candida albicans depends on a functional beta-oxidation pathway: evidence for reduced metabolite transport across the peroxisomal membrane.

The glyoxylate cycle, a metabolic pathway required for generating C(4) units from C(2) compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcellular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5Delta/Delta strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2Delta/Delta strain that is completely deficient in fatty acid beta-oxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2Delta/Delta strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2Delta/Delta strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2Delta/Delta mutant.