TORC1 specifically inhibits microautophagy through ESCRT-0

Abstract

Nutrient starvation induces the degradation of specific plasma membrane proteins through the multivesicular body (MVB) sorting pathway and of vacuolar membrane proteins through microautophagy. Both of these processes require the gateway protein Vps27, which recognizes ubiquitinated cargo proteins at phosphatidylinositol 3-phosphate-rich membranes as part of a heterodimeric complex coined endosomal sorting complex required for transport 0. The target of rapamycin complex 1 (TORC1), a nutrient-activated central regulator of cell growth, directly phosphorylates Vps27 to antagonize its function in microautophagy, but whether this also serves to restrain MVB sorting at endosomes is still an open question. Here, we show that TORC1 inhibits both the MVB pathway-driven turnover of the plasma membrane-resident high-affinity methionine permease Mup1 and the inositol transporter Itr1 and the microautophagy-dependent degradation of the vacuolar membrane-associated v-ATPase subunit Vph1. Using a Vps277D variant that mimics the TORC1-phosphorylated state of Vps27, we further show that cargo sorting of Vph1 at the vacuolar membrane, but not of Mup1 and Itr1 at endosomes, is sensitive to the TORC1-controlled modifications of Vps27. Thus, TORC1 specifically modulates microautophagy through phosphorylation of Vps27, but controls MVB sorting through alternative mechanisms.

Lysosome biology in autophagy

“…The ER is taken up during ER stress 133 . Studies with yeast have also determined that microautophagy is mediated by the ESCRT machinery 134 and regulated by TORC1 activity 135 . The importance of GTP availability, membrane fluidity and membrane potential was also discovered, pointing to the existence of unidentified factors 136 .…”

TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles

“…However, it was recently shown that a second population of endosomes exists, which contains an endosomal pool of TORC1 and the EGO complex. 52 As Ivy1 also strongly colocalizes with EGO components in dots proximal to the vacuole, 49 we wondered if all proteins, including TORC1 and Fab1, function together on signaling endosomes.…”

“…To determine if Fab1 phosphorylation specifically affects TORC1 activity on endosomes or vacuoles ( Figure 4C), we used established TORC1 activity reporters that specifically localize to endosomes (ET) or to vacuoles (VT) 47,52 (see STAR Methods; Figure 4D). We determined TORC1 activity by decorating blots with a phospho-specific antibody to Sch9, which recognizes phosphorylated Sch9-Thr 737 , a bona fide TORC1 target residue in yeast ( Figure 4C).…”

Selective Microautophagy of Proteasomes is Initiated by ESCRT-0 and Is Promoted by Proteasome Ubiquitylation

“…2C, D). In cells grown in rich medium, WT yEGFP-Vps27 concentrated in small foci or granules, which are assumed to be endosomes (Hatakeyama et al, 2019;Katzmann et al, 2003), whereas the mutant derivative displayed a more diffuse pattern, which correlates with cargo protein-sorting defects (Ren and Hurley, 2010). In low glucose, WT yEGFP-Vps27 did not accumulate appreciably within cytoplasmic granules even in the presence of MG132; proteasome inactivation slightly reduced vps27-VHS-UIM granule staining (Fig.…”

“…The membranes were incubated with the following primary antibodies: rabbit anti-Vps27 (Hatakeyama and De Virgilio, 2019) at 1:1000 dilution; JL-8 anti-GFP monoclonal antibody (TaKaRa, catalog # 632381, lot # A8034133) at 1:2,000 dilution; or an anti-Pgk1 monoclonal antibody (Abcam, catalog # ab113687, lot # GR3373682-5) at 1:10,000 dilution. Primary antibody binding was followed by anti-mouse (GE Healthcare, catalog # NXA931V, lot # 17041890) or anti-rabbit IgG (GE Healthcare, catalog # NA934V, lot # 17136627) secondary antibody conjugated to horseradish peroxidase at 1:10,000 dilution.…”