Cysteine-dependent ubiquitination of Pex18p is linked to cargo translocation across the peroxisomal membrane.
The peroxisomal matrix protein import is facilitated by cycling receptor molecules that shuttle between the cytosol and the peroxisomal membrane. In the yeast Saccharomyces cerevisiae, the import of proteins harboring a peroxisomal targeting signal of type II (PTS2) is mediated by the receptor Pex7p and its co-receptor Pex18p. Here we demonstrate that Pex18p undergoes two kinds of ubiquitin modifications. One of these ubiquitination events depends on lysines 13 and 20 and forces rapid Pex18p turnover by proteasomal degradation. A cysteine residue near the extreme Pex18p amino-terminus is required for the second type of ubiquitination. It turned out that this cysteine residue at position 6 is essential for the function of Pex18p in peroxisomal protein import but does not contribute to receptor-cargo association and binding to the peroxisomal import apparatus. However, in contrast to the wild-type protein, cysteine 6-mutated Pex18p is arrested in a membrane-protected state, whereas Pex7p is accessible in a protease protection assay. This finding indicates that Pex18p export is linked to cargo translocation, which supports the idea of an export-driven import of proteins into peroxisomes.
Abteilung für Systembiochemie, Institut für Physiologische Chemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Astrid
A
Hensel
Sabrina
S
Beck
Fouzi
F
El Magraoui
Harald W
HW
Platta
Wolfgang
W
Girzalsky
Ralf
R
Erdmann
D003545:Cysteine / Q000378:metabolism*; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology*
D026901:Membrane Transport Proteins; C116845:PEX18 protein, S cerevisiae; C499509:PEX5 protein, S cerevisiae; C082902:PEX7 protein, S cerevisiae; D000074437:Peroxisomal Targeting Signal 2 Receptor; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D008239:Lysine; D003545:Cysteine