Abstract
Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism. This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation.Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism. Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-linked Ub chains.
Highlights
Peroxisomal matrix protein import in yeast requires the ubiquitin-conjugating enzyme (UBC or E2) Pex4p [1]
In addition to a role in Pex5p ubiquitination, we previously demonstrated that Pex22p from the yeast Saccharomyces cerevisiae (Sc) allows ScPex4p to build lysine 48 (K48)-linked Ub chains in vitro, in an E3 ligase-independent manner [11,12]
The structure of Pex4p alone was solved by molecular replacement using MOLREP [18], using the coordinates of ScPex4p 2y9m as search model, while the structure of the Pex4p-Pex22S complex was solved using a model built from the structure of Pex4p together with ScPex22S from the ScPex4p-Pex22S structure (2y9m)
Summary
Peroxisomal matrix protein import in yeast requires the ubiquitin-conjugating enzyme (UBC or E2) Pex4p [1]. Pex4p, together with a complex consisting of the RING E3 ligases Pex2p, Pex10p and Pex12p, ubiquitinates the receptor proteins Pex5p [2,3] and members of the Pex20p co-receptor family [4,5]. This modification, which occurs on a conserved cysteine residue in Pex5p/ Pex proteins [5,6], facilitates receptor recycling from the peroxisomal membrane [7,8]. Reports showed that Pex22p binding is required for Pex4p to ubiquitinate Pex5p [10,11], possibly for positioning the active site of Pex4p close to the cysteine in Pex5p, to allow ubiquitin (Ub) transfer [11]
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