A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p.
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 NH(2)-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 NH(2)-terminal alpha-NH(2) 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 beta-mercaptoethanol, a compound that is unable to break ubiquitin-NH(2) group linkages. Together, our results strongly suggest that Pex4p-dependent ubiquitination of Pex5p occurs on a cysteine residue.
Williams
Chris
C
Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
van den Berg
Marlene
M
Sprenger
Richard R
RR
Distel
Ben
B
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008623:Mercaptoethanol / Q000737:chemistry; D008969:Molecular Sequence Data; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism
D026901:Membrane Transport Proteins; C076587:PEX4 protein, S cerevisiae; C499509:PEX5 protein, S cerevisiae; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D008623:Mercaptoethanol; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008239:Lysine; D003545:Cysteine