Abstract
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 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 non-phosphorylatable 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 in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.
Highlights
Peroxisomes are highly dynamic metabolic organelles that are present in most eukaryotic cells and fulfill a wide variety of metabolic functions, depending on organism, tissue, and environmental condition
To establish a most comprehensive Pex14p in vivo phosphorylation map, we affinity-purified TEV protease cleavage site-Protein A (TPA)-tagged Pex14p expressed from its native chromosomal location from a crude membrane fraction of yeast cells grown under peroxisome-proliferating conditions (Veenhuis et al, 1987)
The obtained eluate fraction was analyzed by phosphate-affinity (Phos-tag) SDS-PAGE, in which phosphorylated proteins migrate slower than their non-phosphorylated counterparts (Kinoshita et al, FIGURE 1 | Pex14p is multiply phosphorylated in vivo. (A) TPA-tagged Pex14p (Pex14pTPA) expressed from its native chromosomal location was affinity-purified from a Triton X-100-solubilized crude membrane fraction of yeast cells grown in oleic acid-containing medium
Summary
Peroxisomes are highly dynamic metabolic organelles that are present in most eukaryotic cells and fulfill a wide variety of metabolic functions, depending on organism, tissue, and environmental condition. Maintenance of the highly dynamic peroxisomal system and its adjustment to varying physiological conditions rely on several processes including peroxisomal protein targeting, import of matrix proteins and peroxisomal membrane biogenesis proteins as well as growth and division, and turnover of peroxisomes by autophagy. All these processes need to be well coordinated and tightly regulated to ensure the functionality of the peroxisomal system. Since peroxisomes do not contain their own DNA, all peroxisomal matrix proteins are synthesized in the cytosol and need to be imported into the organelle (Walter and Erdmann, 2019). At the end of the import cycle, the receptor is extracted from the peroxisomal membrane in sequential ATP- and ubiquitin-dependent steps
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