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.

Role of PEX5 ubiquitination in maintaining peroxisome dynamics and homeostasis

“…78 Its association with Pex22 stimulates the full E2 activity of Pex4. 79 Pex2, Pex10, and Pex12 form a sub-complex at the peroxisome membrane and regulate the stabilities of each other. 80,81 These three E3 ligases function cooperatively to ubiquitinate Pex5 at the conserved cysteine.…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…Arabidopsis PEX22 was identified via its PEX4-binding ability and can function in yeast when expressed together with Arabidopsis PEX4 (Zolman et al, 2005). Yeast PEX22 enhances PEX4 enzymatic activity (El Magraoui et al, 2014), and a T-DNA insertion upstream of the Arabidopsis PEX22 start codon exacerbates the peroxisomal defects of pex4-1 (Zolman et al, 2005).…”

“…PEX26 binds PEX1-PEX6 via PEX6 (Birschmann et al, 2003;Matsumoto et al, 2003;Goto et al, 2011). Unlike PEX22 enhancement of PEX4 activity (El Magraoui et al, 2014), tether binding decreases PEX1-PEX6 ATPase activity in yeast (Gardner et al, 2015). In addition to tethering PEX1-PEX6, mammalian PEX26 interacts with the PEX14 docking peroxin (Tamura et al, 2014), hinting that PEX26 may bridge the import and export machinery.…”

The Pex4p–Pex22p complex fromHansenula polymorpha: biophysical analysis, crystallization and X-ray diffraction characterization

“…The receptor-recycling step involves monoubiquitination of Pex5p at the conserved cysteine residue (Williams et al, 2007;Okumoto et al, 2011) and requires the action of a number of peroxins, including Pex1p, Pex4p, Pex6p, Pex15p and Pex22p (Koller et al, 1999;Collins et al, 2000;Platta et al, 2008;Rosenkranz et al, 2006). Several studies have shown that the ubiquitin-conjugating (E2) enzyme Pex4p is responsible for Pex5p monoubiquitination in the yeast Saccharomyces cerevisiae (El Magraoui et al, 2014;Williams et al, 2007;Platta et al, 2007). Pex4p associates with the peroxisomal membrane via its interaction with the membrane-bound Pex22p (Koller et al, 1999).…”

“…Pex4p associates with the peroxisomal membrane via its interaction with the membrane-bound Pex22p (Koller et al, 1999). However, Pex22p also acts as a co-activator of Pex4p, stimulating the E2 activity of Pex4p through an unknown mechanism (Williams et al, 2012(Williams et al, , 2013El Magraoui et al, 2014).…”