Pex14p phosphorylation modulates import of citrate synthase 2 into peroxisomes in Saccharomyces cerevisiae

AbstractThe peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and nonphosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes on oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Our data point to the control of the peroxisomal import of Cit2p via the state of Pex14p phosphorylation at S266, which may help S. cerevisiae cells to rapidly adjust their carbohydrate metabolism according to the nutritional conditions.

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Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1399-1405.1990 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1399-1405

Author(s):

A S Lewin ◽

V Hines ◽

G M Small

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Citrate Synthase ◽

Glyoxylate Cycle ◽

Mitochondrial Fraction ◽

Yeast Cells ◽

Mitochondrial Activity ◽

Major Peak ◽

Gene Encoding ◽

Peroxisomal Targeting

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.

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Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1399 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1399-1405 ◽

Cited By ~ 77

Author(s):

A S Lewin ◽

V Hines ◽

G M Small

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Citrate Synthase ◽

Glyoxylate Cycle ◽

Mitochondrial Fraction ◽

Yeast Cells ◽

Mitochondrial Activity ◽

Major Peak ◽

Gene Encoding ◽

Peroxisomal Targeting

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TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import

The Journal of Cell Biology ◽

10.1083/jcb.201611170 ◽

2017 ◽

Vol 216 (9) ◽

pp. 2843-2858 ◽

Cited By ~ 27

Author(s):

Wei Wang ◽

Zhi-Jie Xia ◽

Jean-Claude Farré ◽

Suresh Subramani

Keyword(s):

Oxidative Stress ◽

Amino Acids ◽

Matrix Protein ◽

Protein Import ◽

Gene Mutations ◽

Mulibrey Nanism ◽

Peroxisomal Biogenesis ◽

Peroxisomal Targeting ◽

Targeting Signals ◽

Peroxisomal Biogenesis Disorders

Most proteins destined for the peroxisomal matrix depend on the peroxisomal targeting signals (PTSs), which require the PTS receptor PEX5, whose deficiency causes fatal human peroxisomal biogenesis disorders (PBDs). TRIM37 gene mutations cause muscle–liver–brain–eye (mulibrey) nanism. We found that TRIM37 localizes in peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with its C-terminal 51 amino acids (CT51), which is required for PTS protein import. PEX5 mutations (K464A or ΔCT51), or TRIM37 depletion or mutation, reduce PEX5 abundance by promoting its proteasomal degradation, thereby impairing its functions in cargo binding and PTS protein import in human cells. TRIM37 or PEX5 depletion induces apoptosis and enhances sensitivity to oxidative stress, underscoring the cellular requirement for functional peroxisomes. Therefore, TRIM37-mediated ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import, suggesting that mulibrey nanism is a new PBD.

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Metabolic control of peroxisome abundance

Journal of Cell Science ◽

10.1242/jcs.112.10.1579 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1579-1590 ◽

Cited By ~ 6

Author(s):

C.C. Chang ◽

S. South ◽

D. Warren ◽

J. Jones ◽

A.B. Moser ◽

...

Keyword(s):

Metabolic Control ◽

Matrix Protein ◽

Protein Import ◽

Zellweger Syndrome ◽

Mutant Gene ◽

Peroxisomal Membrane ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Complementation Groups ◽

Peroxisomal Targeting Signal

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

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The Peroxisomal PTS1-Import Defect of PEX1- Deficient Cells Is Independent of Pexophagy in Saccharomyces cerevisiae

International Journal of Molecular Sciences ◽

10.3390/ijms21030867 ◽

2020 ◽

Vol 21 (3) ◽

pp. 867 ◽

Cited By ~ 1

Author(s):

Thomas Mastalski ◽

Rebecca Brinkmeier ◽

Harald W. Platta

Keyword(s):

Autosomal Recessive ◽

Matrix Protein ◽

Protein Import ◽

Beta Oxidation ◽

Working Models ◽

Peroxisomal Biogenesis ◽

Physiologic Role ◽

Peroxisomal Biogenesis Disorders ◽

Peroxisomal Function

The important physiologic role of peroxisomes is shown by the occurrence of peroxisomal biogenesis disorders (PBDs) in humans. This spectrum of autosomal recessive metabolic disorders is characterized by defective peroxisome assembly and impaired peroxisomal functions. PBDs are caused by mutations in the peroxisomal biogenesis factors, which are required for the correct compartmentalization of peroxisomal matrix enzymes. Recent work from patient cells that contain the Pex1(G843D) point mutant suggested that the inhibition of the lysosome, and therefore the block of pexophagy, was beneficial for peroxisomal function. The resulting working model proposed that Pex1 may not be essential for matrix protein import at all, but rather for the prevention of pexophagy. Thus, the observed matrix protein import defect would not be caused by a lack of Pex1 activity, but rather by enhanced removal of peroxisomal membranes via pexophagy. In the present study, we can show that the specific block of PEX1 deletion-induced pexophagy does not restore peroxisomal matrix protein import or the peroxisomal function in beta-oxidation in yeast. Therefore, we conclude that Pex1 is directly and essentially involved in peroxisomal matrix protein import, and that the PEX1 deletion-induced pexophagy is not responsible for the defect in peroxisomal function. In order to point out the conserved mechanism, we discuss our findings in the context of the working models of peroxisomal biogenesis and pexophagy in yeasts and mammals.

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Metabolic and Developmental Effects Resulting from Deletion of the citA Gene Encoding Citrate Synthase in Aspergillus nidulans

Eukaryotic Cell ◽

10.1128/ec.00373-09 ◽

2010 ◽

Vol 9 (4) ◽

pp. 656-666 ◽

Cited By ~ 9

Author(s):

Sandra L. Murray ◽

Michael J. Hynes

Keyword(s):

Aspergillus Nidulans ◽

Fluorescent Protein ◽

Citrate Synthase ◽

Glyoxylate Cycle ◽

Single Gene ◽

Carbon Sources ◽

Tca Cycle ◽

Poor Growth ◽

Content Type ◽

The Glyoxylate Cycle

ABSTRACT Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsAΔ citAΔ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.

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The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins.

The Journal of Cell Biology ◽

10.1083/jcb.135.1.97 ◽

1996 ◽

Vol 135 (1) ◽

pp. 97-109 ◽

Cited By ~ 163

Author(s):

Y Elgersma ◽

L Kwast ◽

A Klein ◽

T Voorn-Brouwer ◽

M van den Berg ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Protein ◽

Protein Import ◽

Sh3 Domain ◽

Type I ◽

Peroxisomal Membrane ◽

Peroxisomal Membrane Protein ◽

Peroxisomal Targeting ◽

Src Homology

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.

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Functional Similarity between the Peroxisomal PTS2 Receptor Binding Protein Pex18p and the N-Terminal Half of the PTS1 Receptor Pex5p

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.8895-8906.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 8895-8906 ◽

Cited By ~ 74

Author(s):

Antje Schäfer ◽

Daniela Kerssen ◽

Marten Veenhuis ◽

Wolf-H. Kunau ◽

Wolfgang Schliebs

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Receptor Binding ◽

Matrix Protein ◽

Protein Import ◽

Chimeric Protein ◽

Functional Similarity ◽

Cargo Transport ◽

Receptor Binding Protein ◽

Import Receptor

ABSTRACT Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.

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The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20

Molecular Biology of the Cell ◽

10.1091/mbc.e13-12-0716 ◽

2014 ◽

Vol 25 (17) ◽

pp. 2634-2643 ◽

Cited By ~ 11

Author(s):

Danielle Hagstrom ◽

Changle Ma ◽

Soumi Guha-Polley ◽

Suresh Subramani

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Degradation Pathway ◽

Matrix Proteins ◽

Receptor Recycling ◽

Wild Type ◽

The Matrix ◽

Peroxisomal Targeting ◽

Targeting Signals ◽

The Stability

Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways.

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