Grou Cláudia P, Carvalho Andreia F, Pinto Manuel P, Huybrechts Sofie J, Sá-Miranda Clara, Fransen Marc, Azevedo Jorge E
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.
J Biol Chem. 2009 Apr 17;284(16):10504-13. doi: 10.1074/jbc.M808978200. Epub 2009 Feb 10.
Pex5p, the peroxisomal protein cycling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During its transient passage through the membrane, Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol. Here we describe the properties of the soluble and membrane-bound monoubiquitinated Pex5p species (Ub-Pex5p). Our data suggest that 1) Ub-Pex5p is deubiquitinated by a combination of context-dependent enzymatic and nonenzymatic mechanisms; 2) soluble Ub-Pex5p retains the capacity to interact with the peroxisomal import machinery in a cargo-dependent manner; and 3) substitution of the conserved cysteine residue of Pex5p by a lysine results in a quite functional protein both in vitro and in vivo. Additionally, we show that MG132, a proteasome inhibitor, blocks the import of a peroxisomal reporter protein in vivo.
过氧化物酶体蛋白循环受体Pex5p在细胞质中结合新合成的过氧化物酶体基质蛋白，并促进它们跨细胞器膜的转运。在其短暂穿过膜的过程中，Pex5p在一个保守的半胱氨酸残基上发生单泛素化，这是其随后依赖ATP返回细胞质所必需的。在此，我们描述了可溶性和膜结合的单泛素化Pex5p物种（Ub-Pex5p）的特性。我们的数据表明：1）Ub-Pex5p通过上下文依赖的酶促和非酶促机制的组合进行去泛素化；2）可溶性Ub-Pex5p保留了以依赖货物的方式与过氧化物酶体导入机制相互作用的能力；3）将Pex5p的保守半胱氨酸残基替换为赖氨酸会产生一种在体外和体内均具有相当功能的蛋白质。此外，我们表明蛋白酶体抑制剂MG132在体内会阻断过氧化物酶体报告蛋白的导入。
