Pex14p phosphorylation modulates import of citrate synthase 2 into peroxisomes inSaccharomyces cerevisiae

Abstract

<jats:title>Abstract</jats:title><jats:p>The 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 <jats:italic>Saccharomyces cerevisiae</jats:italic>. 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 <jats:italic>S. cerevisiae</jats:italic> cells to rapidly adjust their carbohydrate metabolism according to the nutritional conditions.</jats:p>

Identification of Pex34p as a component of the peroxisomal de novo biogenesis machinery in yeast

“…These phosphatases are conserved among eukaryotes and a peroxisomal localization was reported for the protein from Arabidopsis thaliana (Kataya et al, 2019, Lillo et al, 2014). Recently, regulation of peroxisomal matrix protein import via phosphorylation of Pex14p, a key component of the import machinery, was reported, is may be that yeast Tpd3p in involved in this process (Schummer et al, 2020). Shr5p (Erf4) is involved in the palmitoylation and subcellular localization of yeast Ras protein (Zhao et al, 2002).…”

Versatile allosteric properties in Pex5‐like tetratricopeptide repeat proteins to induce diverse downstream function

“…This binding site appears to be required for the import of oligomeric PTS1 cargos as shown by the loss of import of tetrameric catalase 101 . Finally, DTM phosphorylation may also have an important role as it has been shown that specific Pex14 phosphorylation sites may affect interactions between DTM components, which could also affect the Pex5 export and subsequent recycling process 102‐104 . The function of these phosphorylation sites remains to be determined, and thus far only defects in the import of certain cargos (catalase, citrate synthase 2) under specific conditions such as mitosis and oxidative stress have been detected, rather than a more general effect on cargo import 103,104 …”

Current advances in the function and biogenesis of peroxisomes and their roles in health and disease

“…The talk by Renate Maier (Bettina Warscheid laboratory, University of Freiburg, Germany) addressed the question if phosphorylation of Pex14p plays a role in regulating peroxisomal matrix protein import. They established a comprehensive Pex14p in vivo phosphorylation map of Saccharomyces cerevisiae using high-resolution mass spectrometry (Schummer et al 2020 ). Their analysis of single phosphomimicking and non-phosphorylatable Pex14p variants revealed that a phosphomimetic mutation of Pex14p at serine 266 results in impaired growth and reduced peroxisomal import of citrate synthase 2 (Cit2p).…”