Wang Li, Merz Alexey J, Collins Kevin M, Wickner William
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
J Cell Biol. 2003 Feb 3;160(3):365-74. doi: 10.1083/jcb.200209095.
Vacuole tethering, docking, and fusion proteins assemble into a "vertex ring" around the apposed membranes of tethered vacuoles before catalyzing fusion. Inhibitors of the fusion reaction selectively interrupt protein assembly into the vertex ring, establishing a causal assembly hierarchy: (a) The Rab GTPase Ypt7p mediates vacuole tethering and forms the initial vertex ring, independent of t-SNAREs or actin; (b) F-actin disassembly and GTP-bound Ypt7p direct the localization of other fusion factors; (c) The t-SNAREs Vam3p and Vam7p regulate each other's vertex enrichment, but do not affect Ypt7p localization. The v-SNARE Vti1p is enriched at vertices by a distinct pathway that is independent of the t-SNAREs, whereas both t-SNAREs will localize to vertices when trans-pairing of SNAREs is blocked. Thus, trans-SNARE pairing is not required for SNARE vertex enrichment; and (d) The t-SNAREs regulate the vertex enrichment of both G-actin and the Ypt7p effector complex for homotypic fusion and vacuole protein sorting (HOPS). In accord with this hierarchy concept, the HOPS complex, at the end of the vertex assembly hierarchy, is most enriched at those vertices with abundant Ypt7p, which is at the start of the hierarchy. Our findings provide a unique view of the functional relationships between GTPases, SNAREs, and actin in membrane fusion.
液泡拴系、对接和融合蛋白在催化融合之前，围绕拴系液泡的并列膜组装成一个“顶点环”。融合反应抑制剂选择性地阻断蛋白组装进入顶点环，从而建立了一个因果组装层次结构：(a) Rab GTP酶Ypt7p介导液泡拴系并形成初始顶点环，独立于t-SNARE或肌动蛋白；(b) F-肌动蛋白解聚和结合GTP的Ypt7p指导其他融合因子的定位；(c) t-SNARE Vam3p和Vam7p相互调节对方在顶点的富集，但不影响Ypt7p的定位。v-SNARE Vti1p通过一条独立于t-SNARE的不同途径在顶点富集，而当SNARE的反式配对被阻断时，两种t-SNARE都会定位到顶点。因此，SNARE在顶点的富集不需要反式SNARE配对；(d) t-SNARE调节G-肌动蛋白和Ypt7p效应复合物在顶点的富集，用于同型融合和液泡蛋白分选(HOPS)。与这个层次概念一致，在顶点组装层次结构末端的HOPS复合物，在具有丰富Ypt7p的那些顶点处富集程度最高，而Ypt7p处于层次结构的起始位置。我们的发现为GTP酶、SNARE和肌动蛋白在膜融合中的功能关系提供了独特的见解。
