Abteilung für Systembiochemie, Institut für Physiologische Chemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany.
J Biol Chem. 2011 Dec 16;286(50):43495-505. doi: 10.1074/jbc.M111.286104. Epub 2011 Oct 23.
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.
过氧化物酶体基质蛋白的输入是由在细胞质和过氧化物酶体膜之间循环的受体分子来促进的。在酵母酿酒酵母中，含有 II 型过氧化物酶体靶向信号（PTS2）的蛋白质的输入是由受体 Pex7p 和其共受体 Pex18p 介导的。在这里，我们证明 Pex18p 经历了两种泛素修饰。这些泛素化事件之一依赖于赖氨酸 13 和 20，并通过蛋白酶体降解导致 Pex18p 的快速周转。靠近 Pex18p 氨基末端的极端位置的一个半胱氨酸残基是 Pex18p 在过氧化物酶体蛋白输入中的功能所必需的。事实证明，该位置的 6 号半胱氨酸残基对于 Pex18p 在过氧化物酶体蛋白输入中的功能是必需的，但对受体-货物的结合和与过氧化物酶体输入装置的结合没有贡献。然而，与野生型蛋白相比，半胱氨酸 6 突变的 Pex18p 被阻止在膜保护状态，而 Pex7p 在蛋白酶保护测定中是可接近的。这一发现表明 Pex18p 的输出与货物易位有关，这支持了蛋白质输入过氧化物体的输出驱动的想法。
