title: The cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor.
abstract: Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential.
authors.affiliation: Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum, Germany; Systembiochemie, Ruhr-Universität Bochum, Bochum, Germany; Biomedizinische Forschung, Leibniz-Institut für Analytische Wissenschaften - ISAS -e.V., Dortmund, Germany.
authors.forename: Fouzi
authors.initials: F
authors.lastname: El Magraoui
authors.affiliation: Medizinische Proteomik/Bioanalytik, AG Neuro Proteomics, Medizinisches Proteom-Center, Bochum, Germany; Biomedizinische Forschung, Leibniz-Institut für Analytische Wissenschaften - ISAS -e.V., Dortmund, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schrötter
authors.affiliation: Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Brinkmeier
authors.affiliation: Systembiochemie, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Kunst
authors.affiliation: Medizinische Proteomik/Bioanalytik, AG Neuro Proteomics, Medizinisches Proteom-Center, Bochum, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mastalski
authors.affiliation: Funktionelle Proteomik, Medizinisches Proteom-Center, Bochum, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Müller
authors.affiliation: Funktionelle Proteomik, Medizinisches Proteom-Center, Bochum, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
authors.affiliation: Biomedizinische Forschung, Leibniz-Institut für Analytische Wissenschaften - ISAS -e.V., Dortmund, Germany.
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.affiliation: Systembiochemie, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Girzalsky
authors.affiliation: Systembiochemie, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Erdmann
authors.affiliation: Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Harald W
authors.initials: HW
authors.lastname: Platta
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D008565:Membrane Proteins; C070895:PEX3 protein, S cerevisiae; C076587:PEX4 protein, S cerevisiae; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; C539583:Pex22 protein, S cerevisiae; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C511586:PEX1 protein, S cerevisiae; C490760:PEX6 protein, S cerevisiae