TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy

Abstract

Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the upregulation of autophagy and vacuole proteins involved in recycling, but also result in the downregulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Downregulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

“…AP-3 is likely a substantial contributor to the vacuole membrane proteome, yet relatively few AP-3 cargo proteins have been identified, and none of these are among the 84 hits from our screen. However, because most yeast knockouts contain a secondary mutation in at least 20% of their cell population that strongly affects cell death, typically increasing death , we tested single cellderived colonies/substrains from each knockout for ten confirmed AP-3 cargo proteins: ALP/Pho8, the protein kinase Yck3 (Sun et al, 2004), a vacuole tSNARE Vam3 (Cowles et al, 1997a) and vSNARE Nyv1 (Darsow et al, 1998), amino acid transporters Ypq1-3 (Llinares et al, 2015), a transmembrane component of ESCRT-0 also involved in phagophore formation Atg27 (Segarra et al, 2015), the AP-3 reporter proteins Sna2 (Renard et al, 2010) and Sna4 (Pokrzywa et al, 2009) of unclarified functions, and two candidate AP-3 cargo Cot1 and Zrt3 (Yang et al, 2020). Small scale validation experiments confirmed the genome-wide screen results for all candidates tested with the exception of Δyck3 substrains, which were strikingly death-resistant similar to AP-3 knockouts in the log phase heat-ramp assay from figure 1A (Figure 5F, Supplementary Figure S1 and S4).…”

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids

“…Yet, not only substrate transport can induce nutrient transporter endocytosis, but also nutrient limitation, acute starvation or rapamycin treatment ( Crapeau et al, 2014 ; Hovsepian et al, 2017 ; Jones et al, 2012 ; Laidlaw et al, 2020 ; Lang et al, 2014 ; Müller et al, 2015 ; Schmidt et al, 1998 ; Yang et al, 2020 ). Starvation-induced endocytosis is part of a coordinated catabolic cascade.…”

An ancient RAB5 governs the formation of additional vacuoles and cell shape in petunia petals

“…Vacuolinos do contain ILVs at low numbers and at much higher numbers in enlarged vacuolinos in RAB5a OE cells. It is, however, unclear whether these ILVs originate from MVB precursors or the internalization of vesicles from the vacuolino membrane, by microautophagy, as part of a degradation process (Yang et al, 2020), or both.…”