A Conserved Cysteine Is Essential for Pex4p-dependent Ubiquitination of the Peroxisomal Import Receptor Pex5p

• Published in: The Journal of biological chemistry 2007-08, Vol.282 (31), p.22534-22543
• Main Authors: Williams, Chris, van den Berg, Marlene, Sprenger, Richard R., Distel, Ben
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cysteine - chemistry; Humans; Lysine - chemistry; Membrane Transport Proteins - metabolism; Mercaptoethanol - chemistry; Molecular Sequence Data; Peroxins; Peroxisome-Targeting Signal 1 Receptor; Plasmids - metabolism; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Ubiquitin - chemistry; Ubiquitin - metabolism; Ubiquitin-Conjugating Enzymes - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M702038200

The peroxisomal protein import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and -independent manner. Here we show that the two types of ubiquitination target different residues in the NH2-terminal region of Pex5p and we identify Pex4p (Ubc10p) as the ubiquitin-conjugating enzyme required for Ubc4p-independent ubiquitination. Whereas Ubc4p-dependent ubiquitination occurs on two lysine residues, Pex4p-dependent ubiquitination neither requires lysine residues nor the NH2-terminal α-NH2 group. Instead, a conserved cysteine residue appears to be essential for both the Pex4p-dependent ubiquitination and the overall function of Pex5p. In addition, we show that this form of ubiquitinated Pex5p is susceptible to the reducing agent β-mercaptoethanol, a compound that is unable to break ubiquitin-NH2 group linkages. Together, our results strongly suggest that Pex4p-dependent ubiquitination of Pex5p occurs on a cysteine residue.