TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles

Chen, Zilei; Malia, Pedro Carpio; Hatakeyama, Riko; Nicastro, Raffaele; Hu, Zehan; Péli-Gulli, Marie Pierre; Gao, Jieqiong; Nishimura, Taki; Eskes, Elja; Stefan, Christopher J.; Winderickx, Joris; Dengjel, Jörn; Virgilio, Claudio De; Ungermann, Christian

doi:10.1016/j.cub.2020.10.026

Cited by 41 publications

(91 citation statements)

References 83 publications

(134 reference statements)

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“…We were particularly interested in the Kog1 subunit, as it was one of our most abudant hits in the proteomics screen for Vps501 interactors (Figure 2). Similar to what others have found, we localized Kog1-2XGFP to two independent TORC1 pools; one around the vacuolar membrane and a second in dot-like perivacuolar structures (Chen et al, 2021;Hatakeyama et al, 2019)(Figure 8A). This dual localization is consistently present in wildtype, vps501∆ or sea1∆ cells.…”

Section: The Torc1 Complex and Induction Of Autophagy Are Defective In Vps501∆sea1∆ Cellssupporting

confidence: 88%

“…Lastly, we have defined the role of Vps501 as a co-regulator of autophagy that promotes SEACIT inhibition of TORC1 during autophagy induction. The yeast Rag GTPase-TORC1 complex is found in two spatially and functionally distinct pools, on both the vacuolar and endosomal membranes (Chen et al, 2021;Hatakeyama et al, 2019). Vacuolar TORC1 promotes protein synthesis through its proximal effector Sch9, while endosomal TORC1 controls autophagy induction through phosphorylation of Atg13, preventing Atg1 complex formation at the preautophagosomal structure.…”

Section: Discussionmentioning

confidence: 99%

“…The vacuolar Kog1-2XGFP pool appears be reduced, either in the presence or absence of nitrogen (Figure 8A-B). Recently, these dot-like TORC1 structures have been referred to as signaling endosomes and have been shown to have unique phosphorylation targets (Chen et al, 2021;Hatakeyama et al, 2019). One such target is Atg13, a regulatory subunit of the Atg1 signaling complex that is required for induction of autophagy (Kamada et al, 2000).…”

Section: The Torc1 Complex and Induction Of Autophagy Are Defective In Vps501∆sea1∆ Cellsmentioning

confidence: 99%

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Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to induce autophagy

Goyal¹,

Am³

et al. 2021

Preprint

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The sorting nexins (SNX), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX-BAR proteins, a SNX subfamily characterized by a C-terminal dimeric Bin/Amphiphysin/Rvs (BAR) lipid curvature domain and a conserved Phox-homology domain, are of great interest. In budding yeast, many SNX-BARs proteins have well-characterized endo-vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX-BAR protein, Vps501, with a novel endo-vacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and works with the SEA complex to regulate autophagy. Furthermore, we found cells displayed a severe deficiency in starvation-induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during autophagy. Additionally, we found the SEA complex becomes destabilized in vps501Δsea1Δ cells, which resulted in aberrant TORC1 hyperactivity and misregulation of autophagy induction.

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Section: The Torc1 Complex and Induction Of Autophagy Are Defective In Vps501∆sea1∆ Cellssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: The Torc1 Complex and Induction Of Autophagy Are Defective In Vps501∆sea1∆ Cellsmentioning

confidence: 99%

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Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to induce autophagy

Goyal¹,

Am³

et al. 2021

Preprint

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“…Our results strongly suggest that the protein binds to the vacuole via its N-terminal domain (residues 1-182), while and C2 domain is dispensable (Figures 1 and S1). While this manuscript was in preparation, a study was published that confirmed our observations on the in vivo localization of Sch9 truncation mutants, and showed that the N-terminal domain of Sch9 preferentially interacts in vitro with PI(3,5)P 2 and localizes on the vacuole in vivo (Chen et al , 2021). It will be interesting to elucidate the mechanism of this interaction, since the N-terminal domain of Sch9 does not seem to correspond to any known lipid-binding domain.…”

Section: Discussionsupporting

confidence: 67%

Vacuolar localization via the N-terminal domain of Sch9 is required for TORC1-dependent phosphorylation and downstream signal transduction

Novarina

Guerra

Milias-Argeitis

2021

Preprint

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The budding yeast Sch9 kinase (functional ortholog of the mammalian S6 kinase) is a major effector of the Target of Rapamycin Complex 1 (TORC1) complex in the regulation of cell growth in response to nutrient availability and stress. In budding yeast, Sch9 is partially localized at the vacuolar surface, where it is phosphorylated by TORC1 under favorable growth conditions. Sch9 recruitment at the vacuole is mediated by direct interaction between PI(3,5)P2 on the vacuolar membrane and the region of Sch9 encompassing amino acid residues 1-390, which contains a C2 domain. Since many C2 domains mediate phospholipid binding, it had been suggested that the C2 domain of Sch9 mediates its vacuolar recruitment. However, the in vivo requirement of the C2 domain for Sch9 localization had not been demonstrated, and the phenotypic consequences of Sch9 delocalization remained unknown. Here, by examining cellular localization, phosphorylation state and growth phenotypes of Sch9 truncation mutants, we show that deletion of the N-terminal domain of Sch9 (aa 1-182), but not the C2 domain (aa 183-399), impairs vacuolar localization and TORC1-dependent phosphorylation of Sch9, while causing growth defects similar those observed in sch9Δ cells. Artificial tethering of an N-terminally truncated Sch9 mutant at the vacuolar membrane rescued TORC1-dependent phosphorylation and cell growth. Our study uncovers a key role for the N-terminal domain of Sch9 and demonstrates that recruitment of Sch9 at the vacuolar surface is necessary for TORC1-dependent phosphorylation and downstream signal transduction for the regulation of cell growth.

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“…These results suggest that Fab1 is an upstream component that influences vacuole fragmentation in C. neoformans ( Figure 6 ). In addition to Fab1, TORC1 is also required for vacuole fragmentation in S. cerevisiae [ 5 , 25 ]. Therefore, we tested whether TORC1 also influences vacuole fragmentation in C. neoformans by growing wild-type and sod2 mutant cells with rapamycin.…”

Section: Resultsmentioning

confidence: 99%

Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans

Kim

Song

et al. 2021

JoF

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Vacuoles are dynamic cellular organelles, and their morphology is altered by various stimuli or stresses. Vacuoles play an important role in the physiology and virulence of many fungal pathogens. For example, a Cryptococcus neoformans mutant deficient in vacuolar functions showed significantly reduced expression of virulence factors such as capsule and melanin synthesis and was avirulent in a mouse model of cryptococcosis. In the current study, we found significantly increased vacuolar fragmentation in the C. neoformans mutants lacking SOD1 or SOD2, which respectively encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase. The sod2 mutant showed a greater level of vacuole fragmentation than the sod1 mutant. We also observed that the vacuoles were highly fragmented when wild-type cells were grown in a medium containing high concentrations of iron, copper, or zinc. Moreover, elevated temperature and treatment with the antifungal drug fluconazole caused increased vacuolar fragmentation. These conditions also commonly cause an increase in the levels of intracellular reactive oxygen species in the fungus, suggesting that vacuoles are fragmented in response to oxidative stress. Furthermore, we observed that Sod2 is not only localized in mitochondria but also in the cytoplasm within phagocytosed C. neoformans cells, possibly due to copper or iron limitation.

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TORC1 Determines Fab1 Lipid Kinase Function at Signaling Endosomes and Vacuoles

Cited by 41 publications

References 83 publications

Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to induce autophagy

Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to induce autophagy

Vacuolar localization via the N-terminal domain of Sch9 is required for TORC1-dependent phosphorylation and downstream signal transduction

Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans

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