Cigarette smoking is a risk factor for developing chronic obstructive pulmonary disease and protein aggresome formation is considered to be a hallmark event for the disease. Since dysfunction of lysosome-mediated protein degradation leads to enhanced accumulation of misfolded proteins and subsequent aggresome formation, we examined the effect of cigarette smoke extract (CSE) on ESCRT-mediated sorting in S. cerevisiae as this process is necessary for the functioning of the vacuole, the lysosomal equivalent in yeast. An operational ESCRT pathway is essential for ion homeostasis and our observation that exposure to CSE caused increased sensitivity to LiCl indicated CSE-induced impairment of ESCRT function. To confirm the inhibition of ESCRT function, the targeting of carboxypeptidase S (CPS), which reaches the vacuole lumen via the ESCRT pathway, was examined. Treatment with CSE resulted in the mislocalization of GFP-tagged CPS to the vacuolar membrane, instead of the vacuolar lumen, confirming defective functioning of the ESCRT machinery in CSE-treated cells. Further analysis revealed that CSE-treatment inhibited the recruitment of the ESCRT-0 component, Vps27, to the endosome surface, which is a key event is for the functioning of the ESCRT pathway. This lack of endosomal recruitment of Vps27 most likely results from a depletion of the endosomally-enriched lipid, phosphatidylinositol 3-phosphate (PI3-P), which is the target of Vps27. This is supported by our observation that the presence of excess leucine, a known activator of the lipid kinase responsible for the generation of PI3-P, Vps34, in the medium can rescue the CSE-induced ESCRT misfunctioning. Thus, the current study provides an insight into CSE-induced aggresome formation as it documents that CSE treatment compromises vacuolar degradation due to an impairment of the ESCRT pathway, which likely stems from the inhibition of Vps34. It also indicates that leucine has the potential to attenuate the CSE-induced accumulation of misfolded proteins.

中文翻译：

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香烟烟雾影响酿酒酵母中 ESCRT 介导的液泡活动

吸烟是发展为慢性阻塞性肺病的危险因素，蛋白质聚集体的形成被认为是该疾病的标志性事件。由于溶酶体介导的蛋白质降解功能障碍导致错误折叠蛋白质的积累增加和随后的聚集体形成，我们检查了香烟烟雾提取物 (CSE) 对酿酒酵母中 ESCRT 介导的分选的影响，因为这个过程是必要的液泡，酵母中的溶酶体等价物。可操作的 ESCRT 通路对于离子稳态至关重要，我们观察到暴露于 CSE 会导致对 LiCl 的敏感性增加，这表明 CSE 诱导的 ESCRT 功能受损。为了证实对 ESCRT 功能的抑制，靶向羧肽酶 S (CPS)，它通过 ESCRT 途径到达液泡腔，被检查了。用 CSE 处理导致 GFP 标记的 CPS 错误定位到液泡膜，而不是液泡腔，证实了 CSE 处理细胞中 ESCRT 机制的功能缺陷。进一步的分析表明，CSE 治疗抑制了 ESCRT-0 成分 Vps27 向内体表面的募集，这是 ESCRT 通路功能的关键事件。这种缺乏 Vps27 的内体募集很可能是由于内体富集的脂质、磷脂酰肌醇 3-磷酸 (PI3-P) 耗尽所致，这是 Vps27 的目标。我们观察到过量亮氨酸（一种已知的负责产生 PI3-P、Vps34 的脂质激酶激活剂）的存在可以挽救 CSE 诱导的 ESCRT 功能障碍，这一点得到了支持。因此，目前的研究提供了对 CSE 诱导的聚集体形成的深入了解，因为它记录了 CSE 治疗会由于 ESCRT 途径受损而损害液泡降解，这可能源于 Vps34 的抑制。它还表明亮氨酸有可能减弱 CSE 诱导的错误折叠蛋白质的积累。