Galan J M, Wiederkehr A, Seol J H, Haguenauer-Tsapis R, Deshaies R J, Riezman H, Peter M
Swiss Institute for Experimental Cancer Research, 1066 Epalinges/VD, Switzerland.
Mol Cell Biol. 2001 May;21(9):3105-17. doi: 10.1128/MCB.21.9.3105-3117.2001.
Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin-ligases composed of a core complex including Skp1p, Cdc53p, Hrt1p, the E2 enzyme Cdc34p, and one of multiple F-box proteins which are thought to provide substrate specificity to the complex. Here we show that the F-box protein Rcy1p is required for recycling of the v-SNARE Snc1p in Saccharomyces cerevisiae. Rcy1p localized to areas of polarized growth, and this polarized localization required its CAAX box and an intact actin cytoskeleton. Rcy1p interacted with Skp1p in vivo in an F-box-dependent manner, and both deletion of its F box and loss of Skp1p function impaired recycling. In contrast, cells deficient in Cdc53p, Hrt1p, or Cdc34p did not exhibit recycling defects. Unlike the case for F-box proteins that are known to participate in SCF complexes, degradation of Rcy1p required neither its F box nor functional 26S proteasomes or other SCF core subunits. Importantly, Skp1p was the only major partner that copurified with Rcy1p. Our results thus suggest that a complex composed of Rcy1p and Skp1p but not other SCF components may play a direct role in recycling of internalized proteins.
Skp1p-遍在蛋白连接酶E3（SCF）复合物是一种泛素连接酶，由一个核心复合物组成，该核心复合物包括Skp1p、Cdc53p、Hrt1p、E2酶Cdc34p以及多种F-盒蛋白之一，这些F-盒蛋白被认为赋予该复合物底物特异性。在这里，我们表明F-盒蛋白Rcy1p是酿酒酵母中v-SNARE Snc1p循环利用所必需的。Rcy1p定位于极性生长区域，这种极性定位需要其CAAX框和完整的肌动蛋白细胞骨架。Rcy1p在体内以F-盒依赖的方式与Skp1p相互作用，其F盒的缺失和Skp1p功能的丧失均会损害循环利用。相比之下，缺乏Cdc53p、Hrt1p或Cdc34p的细胞未表现出循环利用缺陷。与已知参与SCF复合物的F-盒蛋白不同，Rcy1p的降解既不需要其F盒，也不需要功能性的26S蛋白酶体或其他SCF核心亚基。重要的是，Skp1p是与Rcy1p共纯化的唯一主要伙伴。因此，我们的结果表明，由Rcy1p和Skp1p而非其他SCF成分组成的复合物可能在内在化蛋白质的循环利用中起直接作用。
