Takeuchi Junko, Chen Hui, Coffino Philip
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USA.
EMBO J. 2007 Jan 10;26(1):123-31. doi: 10.1038/sj.emboj.7601476. Epub 2006 Dec 7.
To determine the minimum requirements for substrate recognition and processing by proteasomes, the functional elements of a ubiquitin-independent degradation tag were dissected. The 37-residue C-terminus of ornithine decarboxylase (cODC) is a native degron, which also functions when appended to diverse proteins. Mutating the cysteine 441 residue within cODC impaired its proteasome association in the context of ornithine decarboxylase and prevented the turnover of GFP-cODC in yeast cells. Degradation of GFP-cODC with C441 mutations was restored by providing an alternate proteasome association element via fusion to the Rpn10 proteasome subunit. However, Rpn10-GFP was stable, unless extended by cODC or other peptides of similar size. In vitro reconstitution experiments confirmed the requirement for both proteasome tethering and a loosely structured region. Therefore, cODC and degradation tags in general must serve two functions: proteasome association and a site, consisting of an extended peptide region, used for initiating insertion into the protease.
为了确定蛋白酶体识别和处理底物的最低要求，对泛素非依赖性降解标签的功能元件进行了剖析。鸟氨酸脱羧酶（cODC）的37个残基的C末端是一个天然降解子，当附加到多种蛋白质上时也能发挥作用。在鸟氨酸脱羧酶的背景下，突变cODC内的半胱氨酸441残基会损害其与蛋白酶体的结合，并阻止酵母细胞中GFP-cODC的周转。通过与Rpn10蛋白酶体亚基融合提供一个替代的蛋白酶体结合元件，可恢复具有C441突变的GFP-cODC的降解。然而，Rpn10-GFP是稳定的，除非通过cODC或其他类似大小的肽进行延伸。体外重建实验证实了蛋白酶体连接和一个结构松散区域的必要性。因此，一般来说，cODC和降解标签必须发挥两种功能：蛋白酶体结合以及一个由延伸肽区域组成的位点，用于启动插入蛋白酶。
