Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
EMBO J. 2013 Feb 20;32(4):552-65. doi: 10.1038/emboj.2012.354. Epub 2013 Jan 11.
Although cellular proteins conjugated to K48-linked Ub chains are targeted to proteasomes, proteins conjugated to K63-ubiquitin chains are directed to lysosomes. However, pure 26S proteasomes bind and degrade K48- and K63-ubiquitinated substrates similarly. Therefore, we investigated why K63-ubiquitinated proteins are not degraded by proteasomes. We show that mammalian cells contain soluble factors that selectively bind to K63 chains and inhibit or prevent their association with proteasomes. Using ubiquitinated proteins as affinity ligands, we found that the main cellular proteins that associate selectively with K63 chains and block their binding to proteasomes are ESCRT0 (Endosomal Sorting Complex Required for Transport) and its components, STAM and Hrs. In vivo, knockdown of ESCRT0 confirmed that it is required to block binding of K63-ubiquitinated molecules to the proteasome. In addition, the Rad23 proteins, especially hHR23B, were found to bind specifically to K48-ubiquitinated proteins and to stimulate proteasome binding. The specificities of these proteins for K48- or K63-ubiquitin chains determine whether a ubiquitinated protein is targeted for proteasomal degradation or delivered instead to the endosomal-lysosomal pathway.
虽然连接到 K48 连接的 Ub 链的细胞蛋白被靶向到蛋白酶体，但连接到 K63-泛素链的蛋白被定向到溶酶体。然而，纯 26S 蛋白酶体以相似的方式结合并降解 K48 和 K63-泛素化的底物。因此，我们研究了为什么 K63-泛素化的蛋白质不能被蛋白酶体降解。我们表明哺乳动物细胞中含有可溶性因子，这些因子选择性地与 K63 链结合，并抑制或阻止它们与蛋白酶体的结合。使用泛素化蛋白作为亲和配体，我们发现与 K63 链选择性结合并阻止其与蛋白酶体结合的主要细胞蛋白是 ESCRT0（内体分选复合物必需的运输）及其成分 STAM 和 Hrs。在体内，ESCRT0 的敲低证实它是阻止 K63-泛素化分子与蛋白酶体结合所必需的。此外，发现 Rad23 蛋白，特别是 hHR23B，特异性结合 K48-泛素化蛋白并刺激蛋白酶体结合。这些蛋白对 K48 或 K63-泛素链的特异性决定了泛素化蛋白是被靶向到蛋白酶体降解还是被递送到内体溶酶体途径。
