Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles-called intracellular vesicle clusters (IVCs)-serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.

中文翻译：

---

细胞内囊泡簇是在酵母中合成细胞外囊泡相关货物蛋白的细胞器。

细胞外囊泡（EVs）在细胞间通讯中起重要作用。在出芽的酵母中（Saccharomyces cerevisiae），EV充当将货物蛋白转运到周质中以在葡萄糖饥饿期间储存的载体。然而，尚未发现合成这些与EV相关的货物蛋白的细胞内细胞器。在这里，我们调查了称为胞内囊泡簇（IVCs）的细胞质细胞器是否可以作为合成目标蛋白的位点，这些蛋白以与EV相关的蛋白的形式分泌。使用蛋白质组学，我们在稳定的低葡萄糖条件下生长的酵母细胞中鉴定了377种IVC相关蛋白，其中最大的一组参与蛋白翻译。分离的IVC显示出需要起始和延伸因子的蛋白质合成活性。我们还确定了隔离的IVC上的431个新合成的蛋白。103Q-GFP是长谷氨酰胺延伸的外源蛋白，它的表达导致该蛋白以大点的形式分布，与IVC标记（包括果糖1,6-双磷酸酶（FBPase）和液泡导入和降解蛋白）共定位Vid24p。我们在用环己酰亚胺处理过的细胞或缺乏IVC形成所需的缺乏VID基因的细胞中未观察到这种模式。在IVC上诱导103Q-GFP不利地影响完整细胞和分离的IVC上的总蛋白合成。该表达还降低了细胞外部分中与EV相关的货物蛋白的水平，而不会影响分泌的EV的数量。