INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352 Jouy-en-Josas, France.
J Proteome Res. 2010 Dec 3;9(12):6169-79. doi: 10.1021/pr100340p. Epub 2010 Nov 12.
The acquisition of the correct folding of membrane proteins is a crucial process that involves several steps from the recognition of nascent protein, its targeting to the endoplasmic reticulum membrane, its insertion, and its sorting to its final destination. Yarrowia lipolytica is a hemiascomycetous dimorphic yeast and an alternative eukaryotic yeast model with an efficient secretion pathway. To better understand the quality control of membrane proteins, we constructed a model system based on the uracil permease. Mutated forms of the permease were stabilized and retained in the cell and made the strains resistant to the 5-fluorouracil drug. To identify proteins involved in the quality control, we separated proteins extracted in nondenaturing conditions on blue native gels to keep proteins associated in complexes. Some gel fragments where the model protein was immunodetected were subjected to mass spectrometry analysis. The proteins identified gave a picture of the folding proteome, from the translocation across the endoplasmic reticulum membrane, the folding of the proteins, to the vesicle transport to Golgi or the degradation via the proteasome. For example, EMC complex, Gsf2p or Yet3p, chaperone membrane proteins of the endoplasmic reticulum were identified in the Y. lipolytica native proteome.
膜蛋白的正确折叠的获得是一个关键的过程，涉及到几个步骤，从识别新生蛋白，其靶向内质网膜，其插入，和它的排序到它的最终目的地。解脂耶氏酵母是一种半子囊菌二相酵母，是一种具有有效分泌途径的替代真核酵母模型。为了更好地理解膜蛋白的质量控制，我们构建了一个基于尿嘧啶通透酶的模型系统。通透酶的突变形式被稳定并保留在细胞中，使菌株对 5-氟尿嘧啶药物产生抗性。为了鉴定参与质量控制的蛋白质，我们在非变性条件下分离蓝 native 凝胶上提取的蛋白质，以保持蛋白质复合物的完整性。一些免疫检测到模型蛋白的凝胶片段被进行质谱分析。鉴定出的蛋白质描绘了折叠蛋白质组，从跨内质网膜的易位、蛋白质的折叠，到囊泡运输到高尔基体或通过蛋白酶体降解。例如，在解脂耶氏酵母天然蛋白质组中鉴定到了 EMC 复合物、Gsf2p 或 Yet3p，它们是内质网膜的伴侣膜蛋白。
