PDF(Pubmed)
Abstract：
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.
摘要：
可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物在膜融合后形成由16层相互作用侧链组成的4螺旋卷曲螺旋束。中央层（层0）是高度保守的，包含三个谷氨酰胺（Q）和一个精氨酸（R），因此SNARE被归类为Qa-，Qb-,QC-,和R-SNAREs。酿酒酵母中的同源液泡融合需要SNAREsVam3（Qa），Vti1(Qb),Vam7(Qc),和Nyv1(R)。然而,缺乏NYV1(nyv1Δ)的酵母菌株没有显示液泡片段化，而vam3Δ和vam7Δ菌株显示出破碎的液泡。这里,我们提供了遗传证据，表明使用新分离的ykt6突变体，R-SNAREsYkt6和Nyv1在体内空泡同型融合中功能冗余。我们观察到ykt6-104突变体在液泡形态上没有缺陷，但是ykt6-104nyv1Δ双突变体具有高度碎片化的液泡。此外，我们显示了由vam7-Q284R突变引起的同型液泡融合的缺陷被nyv1-R192Q或ykt6-R165Q突变所补偿，在SNARE复合物的0层中保持了3Q：1R的比例，表明Nyv1可与液泡SNARE复合体中的Ykt6交换。出乎意料的是,我们发现Ykt6与胞吐Q-SNAREs组装时，内在胞吐R-SNAREsSnc1及其旁系物Snc2失去了组装成胞吐SNARE复合体的能力。这些结果表明，当其他R-SNARE功能失调时，Ykt6可以作为备份，并且SNARE复合物的这种灵活组装可以帮助细胞维持囊泡转运网络的鲁棒性。
