Williams Chris, van den Berg Marlene, Sprenger Richard R, Distel Ben
Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
J Biol Chem. 2007 Aug 3;282(31):22534-43. doi: 10.1074/jbc.M702038200. Epub 2007 Jun 5.
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.
过氧化物酶体蛋白输入受体Pex5p会以依赖和不依赖Ubc4p的方式被泛素修饰。在此我们表明，这两种泛素化作用针对Pex5p氨基末端区域的不同残基，并且我们确定Pex4p（Ubc10p）是不依赖Ubc4p的泛素化所需的泛素结合酶。依赖Ubc4p的泛素化作用发生在两个赖氨酸残基上，而依赖Pex4p的泛素化作用既不需要赖氨酸残基也不需要氨基末端的α-NH₂基团。相反，一个保守的半胱氨酸残基似乎对于依赖Pex4p的泛素化作用和Pex5p的整体功能都至关重要。此外，我们表明这种泛素化形式的Pex5p对还原剂β-巯基乙醇敏感，β-巯基乙醇是一种无法破坏泛素-NH₂基团连接的化合物。总之，我们的结果有力地表明，Pex5p依赖Pex4p的泛素化作用发生在一个半胱氨酸残基上。
