Flierman Dennis, Coleman Catherine S, Pickart Cecile M, Rapoport Tom A, Chau Vincent
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11589-94. doi: 10.1073/pnas.0605215103. Epub 2006 Jul 25.
In cells expressing human cytomegalovirus US11 protein, newly synthesized MHC class I heavy chains (HCs) are rapidly dislocated from the endoplasmic reticulum (ER) and degraded in the cytosol, a process that is similar to ER-associated degradation (ERAD), the pathway used for degradation of misfolded ER proteins. US11-triggered movement of HCs into the cytosol requires polyubiquitination, but it is unknown which ubiquitin-conjugating and ubiquitin-ligase enzymes are involved. To identify the ubiquitin-conjugating enzyme (E2) required for dislocation, we used a permeabilized cell system, in which endogenous cytosol can be replaced by cow liver cytosol. By fractionating the cytosol, we show that E2-25K can serve as the sole E2 required for dislocation of HCs in vitro. Purified recombinant E2-25K, together with components that convert this E2 to the active E2-ubiquitin thiolester form, can substitute for crude cytosol. E2-25K cannot be replaced by the conjugating enzymes HsUbc7/Ube2G2 or Ube2G1, even though HsUbc7/Ube2G2 and its yeast homolog Ubc7p are known to participate in ERAD. The activity of E2-25K, as measured by ubiquitin dimer formation, is strikingly enhanced when added to permeabilized cells, likely by membrane-bound ubiquitin protein ligases. To identify these ligases, we tested RING domains of various ligases for their activation of E2-25K in vitro. We found that RING domains of gp78/AMFR, a ligase previously implicated in ERAD, and MARCHVII/axotrophin, a ligase of unknown function, greatly enhanced the activity of E2-25K. We conclude that in permeabilized, US11-expressing cells polyubiquitination of the HC substrate can be catalyzed by E2-25K, perhaps in cooperation with the ligase MARCHVII/axotrophin.
在表达人巨细胞病毒US11蛋白的细胞中，新合成的MHC I类重链（HCs）会迅速从内质网（ER）错位并在胞质溶胶中降解，这一过程类似于内质网相关降解（ERAD），即用于降解错误折叠的内质网蛋白的途径。US11触发的HCs向胞质溶胶的移动需要多聚泛素化，但尚不清楚哪些泛素结合酶和泛素连接酶参与其中。为了鉴定错位所需的泛素结合酶（E2），我们使用了一种通透细胞系统，其中内源性胞质溶胶可以被牛肝胞质溶胶替代。通过对胞质溶胶进行分级分离，我们发现E2-25K可以作为体外HCs错位所需的唯一E2。纯化的重组E2-25K，连同将该E2转化为活性E2-泛素硫酯形式的成分，可以替代粗制胞质溶胶。即使已知HsUbc7/Ube2G2及其酵母同源物Ubc7p参与ERAD，结合酶HsUbc7/Ube2G2或Ube2G1也不能替代E2-25K。当添加到通透细胞中时，通过泛素二聚体形成测量的E2-25K活性显著增强，这可能是由膜结合的泛素蛋白连接酶引起的。为了鉴定这些连接酶，我们在体外测试了各种连接酶的RING结构域对E2-25K的激活作用。我们发现，先前与ERAD相关的连接酶gp78/AMFR和功能未知的连接酶MARCHVII/轴突营养蛋白的RING结构域极大地增强了E2-25K的活性。我们得出结论，在表达US11的通透细胞中，HC底物的多聚泛素化可能由E2-25K催化，可能与连接酶MARCHVII/轴突营养蛋白协同作用。
