Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093.
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
Proc Natl Acad Sci U S A. 2017 Oct 10;114(41):E8637-E8645. doi: 10.1073/pnas.1708394114. Epub 2017 Sep 25.
Ypt1 and Sec4 are essential Rab GTPases that control the early and late stages of the yeast secretory pathway, respectively. A chimera consisting of Ypt1 with the switch I domain of Sec4, Ypt1-SW1, is efficiently activated in vitro by the Sec4 exchange factor, Sec2. This should lead to its ectopic activation in vivo and thereby disrupt membrane traffic. Nonetheless early studies found that yeast expressing Ypt1-SW1 as the sole copy of exhibit no growth defect. To resolve this conundrum, we have analyzed yeast expressing various levels of Ypt1-SW1 We show that even normal expression of Ypt1-SW1 leads to kinetic transport defects at a late stage of the pathway, with secretory vesicles accumulating near exocytic sites. Higher levels are toxic. Toxicity is suppressed by truncation of Uso1, a vesicle tether required for endoplasmic reticulum-Golgi traffic. The globular head of Uso1 binds to Ypt1 and its coiled-coil tail binds to the Golgi-associated SNARE, Sed5. We propose that when Uso1 is inappropriately recruited to secretory vesicles by Ypt1-SW1, the extended coiled-coil tail blocks docking to the plasma membrane. This putative inhibitory function could serve to increase the fidelity of vesicle docking.
Ypt1 和 Sec4 分别是控制酵母分泌途径早期和晚期阶段的必需 Rab GTPase。由 Sec4 的开关 I 结构域与 Ypt1 组成的嵌合体 Ypt1-SW1，可被 Sec2 这种 Sec4 交换因子在体外有效地激活。这将导致其在体内的异位激活，并因此破坏膜运输。尽管如此，早期的研究发现，表达 Ypt1-SW1 作为 Ypt1 的唯一拷贝的酵母并没有生长缺陷。为了解决这个难题，我们分析了表达各种水平的 Ypt1-SW1 的酵母。我们表明，即使 Ypt1-SW1 的正常表达也会导致途径后期的动力学运输缺陷，分泌小泡在胞吐部位附近积累。更高的水平是有毒的。Uso1 的截断可抑制毒性，Uso1 是内质网-高尔基体运输所必需的囊泡连接蛋白。Uso1 的球状头部与 Ypt1 结合，其卷曲螺旋尾巴与高尔基体相关的 SNARE Sed5 结合。我们提出，当 Uso1 被 Ypt1-SW1 不恰当地募集到分泌小泡时，延伸的卷曲螺旋尾巴会阻止与质膜对接。这种假定的抑制功能可能有助于提高囊泡对接的保真度。
