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Phosphorylation of the receptor protein Pex5p modulates import of PTS1 cargoes into peroxisomesPhosphorylation of the receptor protein Pex5p modulates import of PTS1 cargoes into peroxisomes

ABSTRACT: Peroxisomes are dynamic organelles with vital functions in cellular metabolism and dysfunction of peroxisomes is associated with human diseases. To fulfill their multiple roles, peroxisomes rely on import of nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p-2D) show a decreased peroxisomal import of GFP-SKL. We show that the binding affinity between PTS1-protein and Pex5p-2D is reduced. An in vivo analysis of the effect of the phosphomimicking mutants on all known PTS1-proteins revealed that import of most, but not all, cargo proteins is affected. The physiological effect of the phosphomimetic mutations correlate with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Friedel Drepper

LAB HEAD: Bettina Warscheid

PROVIDER: PXD015676 | Pride | 2023-03-10

REPOSITORIES: Pride

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Phosphorylation of the receptor protein Pex5p modulates import of proteins into peroxisomes.

Fischer Sven S Bürgi Jérôme J Gabay-Maskit Shiran S Maier Renate R Mastalski Thomas T Yifrach Eden E Obarska-Kosinska Agnieszka A Rudowitz Markus M Erdmann Ralf R Platta Harald W HW Wilmanns Matthias M Schuldiner Maya M Zalckvar Einat E Oeljeklaus Silke S Drepper Friedel F Warscheid Bettina B

Biological chemistry 20220921 2-3

Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex ...[more]

PMID: 36122347

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Project description:FOXO transcription factors control cellular formation of reactive oxygen species (ROS), which critically contribute to cell survival and cell death in neuroblastoma. Here, we report that C10orf10, also named M-bM-^@M-^\Decidual Protein induced by Progesterone (DEPP)M-bM-^@M-^], is a direct transcriptional target of FOXO3 in human neuroblastoma. As FOXO3-mediated apoptosis involves a biphasic ROS accumulation, we analyzed cellular ROS levels in DEPP-knockdown cells by live-cell imaging. Knockdown of DEPP prevented the primary and secondary ROS accumulation during FOXO3 activation and attenuates FOXO3-induced apoptosis, whereas its overexpression raises cellular ROS levels and sensitizes to cell death. In neuronal cells, cellular steady state ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that enables protein import into peroxisomes, we analyzed the effect of DEPP on peroxisomal function by measuring the catalase enzyme activity. Catalase activity was reduced by conditional DEPP overexpression and significantly increased in DEPP-knockdown cells. Using live cell imaging and fluorescent peroxisomal and mitochondrial probes we demonstrate that DEPP localizes to peroxisomes and mitochondria in neuroblastoma cells. The combined data indicate that DEPP reduces peroxisomal activity and thereby impairs the cellular ROS detoxification capacity and contributes to death sensitization. SH-EP, NB15 neuroblastoma cells and CCRF-CEM-C7H2 acute lymphoblastic leukemia cells were infected with the retrovirus plasmid pLIB-FOXO3(A3)-Ertm-iresNeo. Gene expression measures of samples with activated FOXO3 transcription factor (3h OHT treated) have been compared to untreated samples (0h time point). To rule out gene regulations by estrogen samples treated for 3 hours with tamoxifen have been compared to the untreated samples. Only genes that were more than two-fold regulated in the first, but not in the second comparison were defined to be FOXO3 regulated.

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