TOR complex 2–regulated protein kinase Ypk1 controls sterol distribution by inhibiting StARkin domain–containing proteins located at plasma membrane–endoplasmic reticulum contact sites

Roelants, Françoise M.; Chauhan, Neha; Muir, Alexander; Davis, Jameson C.; Menon, Anant K.; Levine, Tim P.; Thorner, Jeremy

doi:10.1091/mbc.e18-04-0229

Cited by 29 publications

(38 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For yeast cell growth, enlargement of the cell wall needs to be tightly coupled to both an increase in cell mass and expansion of the PM. TORC2-Ypk1 signaling controls the processes that maintain adequate PM levels of all of the lipid classes (sphingolipids, glycerolipids, and sterols) needed to sustain growth and viability (Roelants et al 2017a(Roelants et al , 2018. Thus, down-regulation of TORC2 activity upon CWI pathway activation provides a mechanism by which the status of the cell wall can be sensed by TORC2 and thereby the rates of the reactions necessary for PM homeostasis can be adjusted accordingly.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

Leskoske

Roelants

Emmerstorfer-Augustin

et al. 2018

Genes Dev.

Self Cite

View full text Add to dashboard Cite

Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogenactivated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.

show abstract

Section: Discussionmentioning

confidence: 99%

Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

Leskoske

Roelants

Emmerstorfer-Augustin

et al. 2018

Genes Dev.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cells with aberrations in their PM resulting from sterol and/or sphingolipid dysregulation would be expected to be either sensitive or resistant to these drugs as compared with wild-type cells. Thus, we tested the sensitivity of deletion mutants of the nine uncharacterized genes to nystatin and myriocin, using wild-type and nystatin-and myriocin-sensitive ysp2 cells (Roelants et al, 2018) as controls. As shown in Figure 5a, the deletion of YGL159W, OPY1, F I G U R E 5 Nystatin and myriocin sensitivity of cells lacking functional sterolbinding protein candidate proteins.…”

Section: Phenotypic Analyses Of the Prioritized Uncharacterized Orfsmentioning

confidence: 99%

A PhotoClick cholesterol‐based quantitative proteomics screen for cytoplasmic sterol‐binding proteins in Saccharomyces cerevisiae

Chauhan¹,

Sere²,

Sokòl

et al. 2020

Yeast

Self Cite

View full text Add to dashboard Cite

Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins.Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional-as yet unidentified-sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs.We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking. K E Y W O R D Sclick chemistry, membrane, non-vesicular transport, proteomics, sterol

show abstract

“…Moreover, depletion of both, NHR-8 and SBP-1, mimicked the induced UPR mt observed in sgk-1 loss-of-function mutants (Figure 3D). Apart from sterol [24], ceramide and sphingolipids are also key structural lipids of membranes that are regulated by Ypk1/SGK-1 in yeast [23, 26, 74, 75] and regulate membrane trafficking in C. elegans [76]. We targeted by RNAi sptl-1, a serine palmitoyl-CoA acyltransferase responsible for the first committed step in de novo sphingolipid synthesis, and cgt-3, a ceramide glucosyltransferase, previously shown to interact with SGK-1 [62].…”

Section: Resultsmentioning

confidence: 99%

“…TORC2/Ypk1 activates sphingolipid and ceramide biosynthesis [23, 74]. Furthermore, sphingolipids levels modulate Ypk1 expression in a positive feedback loop to respond to membrane stress [75] and to regulate cell growth [24, 25]. In C. elegans SGK-1 has been proposed to regulate membrane trafficking through sphingolipids [62].…”

Section: Discussionmentioning

confidence: 99%

“…Further, our transcription factor RNAi screen uncovered the implication of membrane sterol and lipid homeostasis in the UPR mt induction triggered by SGK-1 deficiency (Figure 3B). Particularly interesting were NHR-8 and SBP-1/SREBP-1, considering the role of Ypk1 in sphingolipid metabolism [23, 26, 74, 75] and membrane sterol distribution at plasma membrane-endoplasmic reticulum contact sites [24]. Cholesterol and sphingolipids are essential components of cell membranes and are essential for membrane dynamics in the cell.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Prohibitin depletion extends lifespan of a TORC2/SGK-1 mutant through autophagy

Hernando‐Rodríguez

Pérez-Jiménez

Rodríguez-Palero

et al. 2019

Preprint

View full text Add to dashboard Cite

13 14 -2 -Mitochondrial prohibitins (PHB) are highly conserved proteins with a peculiar 15 effect on lifespan. While PHB depletion shortens lifespan of wild type animals, it 16 enhances longevity of a plethora of metabolically compromised mutants, 17 including target of rapamycin complex 2 (TORC2) mutants sgk-1 and rict-1. Here, 18 we show that sgk-1 mutants have impaired mitochondrial homeostasis, 19 lipogenesis, yolk formation and autophagy flux due to alterations in membrane 20 lipid and sterol homeostasis. Remarkably, all these features are suppressed by 21 PHB depletion. Lifespan analysis shows that autophagy and the mitochondrial 22 unfolded protein response (UPR mt ), but not mitophagy, are required for the 23 enhanced longevity caused by PHB depletion in sgk-1 mutants. We hypothesize 24 that UPR mt induction upon PHB depletion extends lifespan of sgk-1 mutants 25 through autophagy. Our results strongly suggest that PHB depletion suppresses 26 the autophagy defects of sgk-1 mutants by altering membrane lipid composition 27 at ER-mitochondria contact sites, where TORC2 localizes. 28 29 93 mitochondria-associated endoplasmic reticulum (ER) membranes (MAM).94 95 -6 - Results 96Prohibitin depletion suppresses the altered mitochondrial structure and 97 function of sgk-1 mutants 98In C. elegans, loss of function of SGK-1 causes a delay in development (Jones et al., 992009) as well as reduced brood and body size (Soukas et al., 2009). These phenotypes 100 are consistent with phenotypes observed in mitochondrial mutants (Dillin et al., 2002). In 101 addition, worms lacking the TORC2 component RICT-1 or the downstream kinase SGK-102 1, have an induced mitochondrial unfolded protein response (UPR mt ) (Gatsi et al., 2014). 103We analyzed whether mitophagy, another mitochondrial quality control mechanism, 104 might be activated in sgk-1 deletion mutants using a mitophagy reporter based on PINK-105 1 protein (Kirienko et al., 2015). PINK-1 is a serine threonine protein kinase that, when 106 mitochondria are depolarized, accumulates in the outer mitochondrial membrane and 107 targets mitochondria for selective autophagy (Narendra and Youle, 2011; Palikaras et 108 al., 2015). We observed that depletion of sgk-1 increased PINK-1 protein levels ( Figure 109 1A). Similarly, PINK-1 protein levels increased upon depletion of phb-1 or atfs-1, the key 110 UPR mt regulator ( Figure 1A), confirming that sgk-1 mutants suffer from mitochondrial 111 stress. 112To better define the mitochondrial defect of sgk-1 deletion mutants we performed 113 transmission electron microscopy (TEM) analysis. Mitochondria in sgk-1 mutants were 114 swollen, and bigger compared to wild type worms in all tissues analyzed (hypodermis, 115 muscle and intestine) at both, day one and day five of adulthood ( Figure 1B and Figure 116 S1A, respectively). In contrast to previous observations (Zhou et al., 2019), no obvious 117 mitochondrial fragmentation or severe cristae defects in the intestine were observed in 118 the TEM sections analyzed. 119-7 -To ...

show abstract

TOR complex 2–regulated protein kinase Ypk1 controls sterol distribution by inhibiting StARkin domain–containing proteins located at plasma membrane–endoplasmic reticulum contact sites

Cited by 29 publications

References 54 publications

Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2

A PhotoClick cholesterol‐based quantitative proteomics screen for cytoplasmic sterol‐binding proteins in Saccharomyces cerevisiae

Prohibitin depletion extends lifespan of a TORC2/SGK-1 mutant through autophagy

Contact Info

Product

Resources

About