ESCRT machinery plays a role in microautophagy in yeast
- PDF / 1,944,967 Bytes
- 10 Pages / 595.276 x 790.866 pts Page_size
- 48 Downloads / 205 Views
(2020) 21:70
BMC Molecular and Cell Biology
RESEARCH ARTICLE
Open Access
ESCRT machinery plays a role in microautophagy in yeast Shamsul Morshed1, Most Naoshia Tasnin1 and Takashi Ushimaru1,2*
Abstract Background: 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 ESCR T in microautophagic processes is required. Results: 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. Conclusions: These findings provide evidence for the direct role of ESCRT in microautophagy induction. Keywords: AP-3 pathway, ESCRT, Microautophagy, Pho8, Vph1, VPS pathway
Background In microautophagy, cytoplasmic cargos are directly engulfed by lysosomal/vacuolar membranes, sorted into the vacuolar lumen, and degraded [1–3]. Because vacuolar membrane proteins together with vacuolar membranes are degraded in the vacuole in the course of microautophagy, overall microautophagic flux is estimated using green fluorescent protein (GFP)-tagged vacuolar transmembrane proteins, Vph1 (a subunit of the vacuolar-ATPase V0 domain) and Pho8 (vacuolar alkaline phosphatase) in the budding yeast Saccharomyces * Correspondence: [email protected] 1 Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8021, Japan 2 Department of Science, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8021, Japan
cerevisiae [4]. When Vph1-GFP and GFP-Pho8 are incorporated into the vacuolar lumen by microautophagy, Vph1 and Pho8, but not the stable GFP moiety, are degraded by vacuolar proteases, generating free GFP, which is detectable by immunoblotting. Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) protein kinase evokes microautophagy [4–6]. The endosomal sorting complex required for transport (ESCRT) system was originally iden
Data Loading...
