ESCRT machinery plays a role in microautophagy in yeast

Shamsul Morshed,Takashi Ushimaru,Most Naoshia Tasnin

doi:10.1186/s12860-020-00314-w

Abstract

BackgroundMicroautophagy, 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.ResultsHere, 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.ConclusionsThese findings provide evidence for the direct role of ESCRT in microautophagy induction.

Highlights

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
Vph1-green fluorescent protein (GFP) is an unsuitable tool to assess the involvement of endosomal sorting complex required for transport (ESCRT) in microautophagy To assess microautophagic flux/activity using a processing assay with a GFP-tagged vacuolar membrane protein, its proper localization in vacuolar membranes is prerequisite
Vph1-GFP did not properly reach the vacuolar surface in ESCRT-deficient cells regardless of TORC1 activity. This demonstrated that the Vph1-GFP processing assay is not suitable to evaluate whether ESCRT is directly implicated in microautophagic processes, Oku et al proposed it based on results obtained using this assay [4]

Summary

Introduction

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. Most proteins destined for vacuoles pass through late endosomes, named the vacuolar protein sorting (VPS) pathway (or the carboxypeptidase Y pathway), and they are trapped in the class E compartment in ESCRT mutants [11, 12] This suggested a possibility that Vph1GFP is not located properly on vacuolar membranes during microautophagy induction, thereby causing a reduction in the autophagic degradation of Vph1-GFP. It remains unclear whether or not ESCRT is directly necessary for microautophagic processes on vacuolar membranes

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