ESCRT machinery is required for proper microautophagy induction after TORC1 inactivation

Microautophagy 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 destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, was properly localized on vacuolar membranes in ESCRT-deficient cells even during microautophagy induction after TORC1 inactivation. Nevertheless, microautophagic degradation of GFP-Pho8 after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation. We confirmed this using another AP-3 pathway-dependent vacuolar membrane protein Sna4. This study provides evidence of the direct role of ESCRT in microautophagy induction.

[1]  T. Ushimaru,et al.  PP2A promotes ESCRT-0 complex formation on vacuolar membranes and microautophagy induction after TORC1 inactivation. , 2020, Biochemical and biophysical research communications.

[2]  T. Ushimaru,et al.  TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast. , 2019, Biochemical and biophysical research communications.

[3]  J. Dengjel,et al.  Spatially Distinct Pools of TORC1 Balance Protein Homeostasis. , 2019, Molecular cell.

[4]  T. Ushimaru,et al.  The TORC1-Nem1/Spo7-Pah1/lipin axis regulates microautophagy induction in budding yeast. , 2018, Biochemical and biophysical research communications.

[5]  Takehiko Kobayashi,et al.  CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy , 2018, The Journal of cell biology.

[6]  Y. Maéda,et al.  Evidence for ESCRT- and clathrin-dependent microautophagy , 2017, The Journal of cell biology.

[7]  J. Hurley,et al.  Membrane budding and scission by the ESCRT machinery: it's all in the neck , 2010, Nature Reviews Molecular Cell Biology.

[8]  E. C. Dell'Angelica,et al.  AP-3-dependent trafficking and disease: the first decade. , 2009, Current opinion in cell biology.

[9]  W. Pokrzywa,et al.  Dual Sorting of the Saccharomyces cerevisiae Vacuolar Protein Sna4p , 2009, Eukaryotic Cell.

[10]  S. Emr,et al.  ESCRTing proteins in the endocytic pathway. , 2007, Trends in biochemical sciences.

[11]  Roger L. Williams,et al.  The emerging shape of the ESCRT machinery , 2007, Nature Reviews Molecular Cell Biology.

[12]  H. Schwarz,et al.  Determination of Four Sequential Stages during Microautophagy in Vitro* , 2004, Journal of Biological Chemistry.

[13]  Linyi Chen,et al.  The yeast casein kinase Yck3p is palmitoylated, then sorted to the vacuolar membrane with AP-3-dependent recognition of a YXXPhi adaptin sorting signal. , 2003, Molecular biology of the cell.

[14]  E. O’Shea,et al.  Global analysis of protein localization in budding yeast , 2003, Nature.

[15]  R. Piper,et al.  Ubiquitin Sorts Proteins into the Intralumenal Degradative Compartment of the Late‐Endosome/Vacuole , 2001, Traffic.

[16]  H. Pelham,et al.  Polar transmembrane domains target proteins to the interior of the yeast vacuole. , 2000, Molecular biology of the cell.

[17]  Heinz Schwarz,et al.  Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding. , 2000 .

[18]  Scott D Emr,et al.  The AP-3 Adaptor Complex Is Essential for Cargo-Selective Transport to the Yeast Vacuole , 1997, Cell.

[19]  R. Chapman.  Vacuolar Sorting: Tracking down an elusive receptor , 1994, Current Biology.

[20]  T. Stevens,et al.  An MBoC Favorite: Morphological classification of the yeast vacuolar protein-sorting mutants: evidence for a prevacuolar compartment in class E vps mutants , 1992, Molecular biology of the cell.