Schummer Andreas, Maier Renate, Gabay-Maskit Shiran, Hansen Tobias, Mühlhäuser Wignand W D, Suppanz Ida, Fadel Amir, Schuldiner Maya, Girzalsky Wolfgang, Oeljeklaus Silke, Zalckvar Einat, Erdmann Ralf, Warscheid Bettina
Faculty of Biology, Biochemistry and Functional Proteomics, Institute of Biology II, University of Freiburg, Freiburg, Germany.
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Front Cell Dev Biol. 2020 Sep 15;8:549451. doi: 10.3389/fcell.2020.549451. eCollection 2020.
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 . 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 cells to adjust their carbohydrate metabolism according to the nutritional conditions.
过氧化物酶体生物发生因子Pex14p是过氧化物酶体基质蛋白导入机制的重要组成部分。它与Pex13p和Pex17p一起，是酵母中与膜相关的过氧化物酶体对接复合物的一部分，促进载有货物的受体蛋白结合，以便将货物蛋白转运到过氧化物酶体中。此外，Pex14p是过氧化物酶体导入孔的一部分。Pex14p在过氧化物酶体基质蛋白导入过程中的核心作用使其成为蛋白质磷酸化等调控机制的明显靶点。为了探究这种可能性，我们检测了……中Pex14p的磷酸化状态。从溶解的膜中亲和纯化的Pex14p经Phos-tag-SDS-PAGE分析显示Pex14p是一种多磷酸化蛋白。通过质谱分析，我们鉴定出16个磷酸化位点，磷酸化热点位于Pex14p的N端和C端区域。对Pex14p的磷酸模拟和非磷酸化变体的分析表明，携带1型过氧化物酶体靶向信号的绿色荧光蛋白（GFP）的导入减少，这表明Pex14p磷酸化在过氧化物酶体基质蛋白导入中具有功能相关性。我们发现这种效应可归因于Pex14p丝氨酸266位点的磷酸模拟突变（Pex14p-S266D）。我们进一步通过高内涵荧光显微镜筛选了23种天然绿色荧光蛋白标记的过氧化物酶体基质蛋白的亚细胞分布。只有柠檬酸合酶的过氧化物酶体同工型Cit2p在Pex14p-S266D突变体中受到影响，表现为胞质定位增加。Cit2p是乙醛酸循环的一部分，当酵母在非发酵碳源上生长时，乙醛酸循环是产生必需碳水化合物所必需的。Pex14p-S266磷酸化位点突变体在油酸和乙醇中分别在过氧化物酶体和胞质溶胶中形成乙酰辅酶A时表现出相反的生长表型。在油酸中过表达Cit2p挽救了表达Pex14p-S266D的酵母细胞的生长表型。我们的数据表明，Pex14p在S266位点的磷酸化提供了一种控制Cit2p过氧化物酶体导入的机制，这有助于细胞根据营养条件调整其碳水化合物代谢。
