Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan.
Department of Science, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan.
BMC Mol Cell Biol. 2020 Oct 7;21(1):70. doi: 10.1186/s12860-020-00314-w.
Microautophagy, which degrades cargos by direct lysosomal/vacuolar engulfment of cytoplasmic cargos, is promoted after nutrient starvation and the inactivation of target of rapamycin complex 1 (TORC1) protein kinase. In budding yeast, microautophagy has been commonly assessed using processing assays with green fluorescent protein (GFP)-tagged vacuolar membrane proteins, such as Vph1 and Pho8. The endosomal sorting complex required for transport (ESCRT) system is proposed to be required for microautophagy, because degradation of vacuolar membrane protein Vph1 was compromised in ESCRT-defective mutants. However, ESCRT is also critical for the vacuolar sorting of most vacuolar proteins, and hence reexamination of the involvement of ESCRT in microautophagic processes is required.
Here, we show that the Vph1-GFP processing assay is unsuitable for estimating the involvement of ESCRT in microautophagy, because Vph1-GFP accumulated highly in the prevacuolar class E compartment in ESCRT mutants. In contrast, GFP-Pho8 and Sna4-GFP destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, were properly localized on vacuolar membranes in ESCRT-deficient cells. Nevertheless, microautophagic degradation of GFP-Pho8 and Sna4-GFP after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation.
These findings provide evidence for the direct role of ESCRT in microautophagy induction.
微自噬通过直接溶酶体/液泡吞噬细胞质货物来降解货物，在营养饥饿和雷帕霉素靶蛋白复合物 1（TORC1）蛋白激酶失活后被促进。在 budding yeast 中，通常使用 GFP 标记的液泡膜蛋白（如 Vph1 和 Pho8）的加工测定来评估微自噬。内体分选复合物必需的运输（ESCRT）系统被认为是微自噬所必需的，因为 ESCRT 缺陷突变体中 Vph1 溶酶体膜蛋白的降解受到损害。然而，ESCRT 对于大多数液泡蛋白的液泡分选也是至关重要的，因此需要重新检查 ESCRT 在微自噬过程中的参与。
在这里，我们表明 Vph1-GFP 处理测定不适合用于估计 ESCRT 在微自噬中的参与，因为 Vph1-GFP 在 ESCRT 突变体中高度积累在前液泡 E 隔室中。相比之下，通过替代衔接蛋白 3（AP-3）途径定位于液泡膜的 GFP-Pho8 和 Sna4-GFP 在 ESCRT 缺陷细胞中正确定位于液泡膜上。然而，在 ESCRT 突变体中，TORC1 失活后 GFP-Pho8 和 Sna4-GFP 的微自噬降解受到阻碍，表明 ESCRT 确实是营养饥饿和 TORC1 失活后微自噬所必需的。
这些发现为 ESCRT 在微自噬诱导中的直接作用提供了证据。
