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

Abstract

Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p-Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p-Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p-Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p-Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p-Pex22p complex and its role in peroxisome biogenesis.

Keywords: Hansenula polymorpha; Pex22; Pex4; peroxisome import; ubiquitin-conjugating enzyme.

Figure 1

Needle-shaped Pex4p crystals produced in 0.1 M MES, 50%(v/v) PEG 200 pH 6.5.

Figure 2

(a) Initial crystals grown in 0.1 M bis-tris propane, 0.2 M ammonium sulfate, 20%(w/v) PEG 3350 pH 7.5 (PACT premier HT-96, Molecular Dimensions). The drops were set up by the sitting-drop method with a Mosquito robot (TTP Labtech) using drops consisting of 160 nl protein solution and 160 nl precipitant solution, and the plates were incubated at 293 K. (b) The final crystals grown in 0.1 M bis-tris propane, 0.2 M ammonium sulfate, 22%(w/v) PEG 3350 pH 7.8. The drops were set up by the sitting-drop method with a Mosquito robot (TTP Labtech) using drops consisting of 160 nl protein solution and 160 nl precipitant solution, and the plates were incubated at 293 K.

Figure 3

MST curve for the binding of Pex4p to Pex22^S, displaying 1:1 stoichiometry. The assay was performed using a fixed concentration of fluorescently labelled Pex4p (10 nM).