WDR41 supports lysosomal response to changes in amino acid availability

C9orf72 mutations are a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. The C9orf72 protein undergoes regulated recruitment to lysosomes and has been broadly implicated in control of lysosome homeostasis. However, although evidence strongly supports an important function for C9orf72 at lysosomes, little is known about the lysosome recruitment mechanism. In this study, we identify an essential role for WDR41, a prominent C9orf72 interacting protein, in C9orf72 lysosome recruitment. Analysis of human WDR41 knockout cells revealed that WDR41 is required for localization of the protein complex containing C9orf72 and SMCR8 to lysosomes. Such lysosome localization increases in response to amino acid starvation but is not dependent on either mTORC1 inhibition or autophagy induction. Furthermore, WDR41 itself exhibits a parallel pattern of regulated association with lysosomes. This WDR41-dependent recruitment of C9orf72 to lysosomes is critical for the ability of lysosomes to support mTORC1 signaling as constitutive targeting of C9orf72 to lysosomes relieves the requirement for WDR41 in mTORC1 activation. Collectively, this study reveals an essential role for WDR41 in supporting the regulated binding of C9orf72 to lysosomes and solidifies the requirement for a larger C9orf72 containing protein complex in coordinating lysosomal responses to changes in amino acid availability.

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C9orf72 binds SMCR8, localizes to lysosomes, and regulates mTORC1 signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e16-01-0003 ◽

2016 ◽

Vol 27 (20) ◽

pp. 3040-3051 ◽

Cited By ~ 96

Author(s):

Joseph Amick ◽

Agnes Roczniak-Ferguson ◽

Shawn M. Ferguson

Keyword(s):

Amino Acid ◽

Structural Similarity ◽

Normal Functions ◽

Availability Analysis ◽

Functional Interactions ◽

Amino Acid Availability ◽

Mtorc1 Signaling ◽

Site Of Action ◽

C9orf72 Gene ◽

Lateral Sclerosis

Hexanucleotide expansion in an intron of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia. However, beyond bioinformatics predictions that suggested structural similarity to folliculin, the Birt-Hogg-Dubé syndrome tumor suppressor, little is known about the normal functions of the C9orf72 protein. To address this problem, we used genome-editing strategies to investigate C9orf72 interactions, subcellular localization, and knockout (KO) phenotypes. We found that C9orf72 robustly interacts with SMCR8 (a protein of previously unknown function). We also observed that C9orf72 localizes to lysosomes and that such localization is negatively regulated by amino acid availability. Analysis of C9orf72 KO, SMCR8 KO, and double-KO cell lines revealed phenotypes that are consistent with a function for C9orf72 at lysosomes. These include abnormally swollen lysosomes in the absence of C9orf72 and impaired responses of mTORC1 signaling to changes in amino acid availability (a lysosome-dependent process) after depletion of either C9orf72 or SMCR8. Collectively these results identify strong physical and functional interactions between C9orf72 and SMCR8 and support a lysosomal site of action for this protein complex.

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p27 regulates the autophagy-lysosomal pathway via the control of Ragulator and mTOR activity in amino acid deprived cells

10.1101/2020.01.07.896860 ◽

2020 ◽

Author(s):

Ada Nowosad ◽

Pauline Jeannot ◽

Caroline Callot ◽

Justine Creff ◽

Renaud T. Perchey ◽

...

Keyword(s):

Amino Acid ◽

Energy Homeostasis ◽

Underlying Mechanism ◽

Mtor Inhibition ◽

Mtorc1 Signaling ◽

And Function ◽

Mtorc1 Activation ◽

Catabolic Process ◽

Mtor Activity ◽

Dependent Mechanism

SummaryAutophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies have shown that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy. However, the underlying mechanism remains unknown. Here, we report that in amino acid deprived cells, p27 controls autophagy via an mTORC1-dependent mechanism. During prolonged amino acid starvation, a fraction of p27 is recruited to lysosomes where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 lysosomal localization and activation. p27 binding to LAMTOR1 prevents Ragulator assembly and function and subsequent mTORC1 activation, thereby promoting autophagy. Conversely, upon amino acid withdrawal, p27−/− cells exhibit elevated mTORC1 signaling, impaired lysosomal activity and autophagy, and resistance to apoptosis. This is associated with sequestration of TFEB in the cytoplasm, preventing the induction of lysosomal genes required for lysosomal function. Silencing of LAMTOR1 or mTOR inhibition restores autophagy and induces apoptosis in p27−/− cells. Together, these results reveal a direct, coordinated regulation between the cell cycle and cell growth machineries.

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Leucine and mTORC1: a complex relationship

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00525.2011 ◽

2012 ◽

Vol 302 (11) ◽

pp. E1329-E1342 ◽

Cited By ~ 141

Author(s):

Kayleigh M. Dodd ◽

Andrew R. Tee

Keyword(s):

Signal Transduction ◽

Protein Synthesis ◽

Amino Acid ◽

Protein Turnover ◽

Muscle Growth ◽

Protein Translation ◽

Amino Acid Availability ◽

Intracellular Amino Acid ◽

System L ◽

Mtorc1 Signaling

Amino acid availability is a rate-limiting factor in the regulation of protein synthesis. When amino acid supplies become restricted, mammalian cells employ homeostatic mechanisms to rapidly inhibit processes such as protein synthesis, which demands high levels of amino acids. Muscle cells in particular are subject to high protein turnover rates to maintain amino acid homeostasis. Mammalian target of rapamycin complex 1 (mTORC1) is an evolutionary conserved multiprotein complex that coordinates a network of signaling cascades and functions as a key mediator of protein translation, gene transcription, and autophagy. Signal transduction through mTORC1, which is centrally involved in muscle growth through enhanced protein translation, is governed by intracellular amino acid supply. The branched-chain amino acid leucine is critical for muscle growth and acts in part through activation of mTORC1. Recent research has revealed that mTORC1 signaling is coordinated primarily at the lysosomal membranes. This discovery has sparked a wealth of research in this field, revealing several different signaling molecules involved in transducing the amino acid signal to mTORC1, including the Rag GTPases, MAP4K3, and Vps34/ULK1. This review evaluates the current knowledge regarding cellular mechanisms that control and sense the intracellular amino acid pool. We discuss the role of leucine and mTORC1 in the regulation of amino acid transport via the system L and system A transporters such as LAT1 and SNAT2, as well as protein degradation via autophagic and proteasomal pathways. We also describe the complexities of energy homeostasis via AMPK and cell receptor-mediated growth signals that also converge on mTORC1. Leucine is a particularly potent regulator of protein turnover, to the extent where leucine stimulation alone is sufficient to stimulate mTORC1 signal transduction. The significance of leucine in this context is not yet known; however, recent advancements in this area will also be covered within this review.

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Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells

Biomolecules ◽

10.3390/biom11091314 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1314

Author(s):

Dan Wang ◽

Xuebin Wan ◽

Xiaoli Du ◽

Zhuxia Zhong ◽

Jian Peng ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Skeletal Muscle Mass ◽

Mammalian Target Of Rapamycin ◽

C2c12 Cells ◽

Amino Acid Transporters ◽

Interacting Proteins ◽

Mtorc1 Signaling ◽

Amino Acid Sensing ◽

Mtorc1 Activation

Amino acids are critical for mammalian target of rapamycin complex 1 (mTORC1) activation on the lysosomal surface. Amino acid transporters SLC38A9 and SLC36A1 are the members of the lysosomal amino acid sensing machinery that activates mTORC1. The current study aims to clarify the interaction of SLC38A9 and SLC36A1. Here, we discovered that leucine increased expressions of SLC38A9 and SLC36A1, leading to mTORC1 activation. SLC38A9 interacted with SLC36A1 and they enhanced each other’s expression levels and locations on the lysosomal surface. Additionally, the interacting proteins of SLC38A9 in C2C12 cells were identified to participate in amino acid sensing mechanism, mTORC1 signaling pathway, and protein synthesis, which provided a resource for future investigations of skeletal muscle mass.

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Gαq activation modulates autophagy by promoting mTORC1 signaling

Nature Communications ◽

10.1038/s41467-021-24811-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sofía Cabezudo ◽

Maria Sanz-Flores ◽

Alvaro Caballero ◽

Inmaculada Tasset ◽

Elena Rebollo ◽

...

Keyword(s):

Nutrient Status ◽

Autophagy Induction ◽

Pathway Activation ◽

Mtorc1 Signaling ◽

Different Types ◽

Mtorc1 Pathway ◽

Mtorc1 Activation ◽

G Protein Coupled ◽

Activation Status

AbstractThe mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.

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PtdIns3P controls mTORC1 signaling through lysosomal positioning

The Journal of Cell Biology ◽

10.1083/jcb.201611073 ◽

2017 ◽

Vol 216 (12) ◽

pp. 4217-4233 ◽

Cited By ~ 55

Author(s):

Zhi Hong ◽

Nina Marie Pedersen ◽

Ling Wang ◽

Maria Lyngaas Torgersen ◽

Harald Stenmark ◽

...

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Transcription Factor ◽

Amino Acid ◽

Cell Periphery ◽

Lipid Kinase ◽

Mechanistic Target Of Rapamycin ◽

Mtorc1 Signaling ◽

Transcription Factor Eb ◽

Mtorc1 Activation

The mechanistic target of rapamycin complex 1 (mTORC1) is a protein kinase complex that localizes to lysosomes to up-regulate anabolic processes and down-regulate autophagy. Although mTORC1 is known to be activated by lysosome positioning and by amino acid–stimulated production of phosphatidylinositol 3-phosphate (PtdIns3P) by the lipid kinase VPS34/PIK3C3, the mechanisms have been elusive. Here we present results that connect these seemingly unrelated pathways for mTORC1 activation. Amino acids stimulate recruitment of the PtdIns3P-binding protein FYCO1 to lysosomes and promote contacts between FYCO1 lysosomes and endoplasmic reticulum that contain the PtdIns3P effector Protrudin. Upon overexpression of Protrudin and FYCO1, mTORC1–positive lysosomes translocate to the cell periphery, thereby facilitating mTORC1 activation. This requires the ability of Protrudin to bind PtdIns3P. Conversely, upon VPS34 inhibition, or depletion of Protrudin or FYCO1, mTORC1-positive lysosomes cluster perinuclearly, accompanied by reduced mTORC1 activity under nutrient-rich conditions. Consequently, the transcription factor EB enters the nucleus, and autophagy is up-regulated. We conclude that PtdIns3P-dependent lysosome translocation to the cell periphery promotes mTORC1 activation.

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Receptor-like role for PQLC2 amino acid transporter in the lysosomal sensing of cationic amino acids

10.1101/2020.07.15.204800 ◽

2020 ◽

Author(s):

Gabriel Talaia ◽

Joseph Amick ◽

Shawn M. Ferguson

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Protein Complex ◽

Underlying Mechanism ◽

Amino Acid Transporter ◽

Cationic Amino Acid ◽

Flexible Loop ◽

Amino Acid Availability ◽

Cationic Amino Acids ◽

Acid Transporter

ABSTRACTPQLC2, a lysosomal cationic amino acid transporter, also serves as a sensor that responds to scarcity of its substrates by recruiting a protein complex comprised of C9orf72, SMCR8 and WDR41 to the surface of lysosomes. This protein complex controls multiple aspects of lysosome function. Although it is known that this response to changes in cationic amino acid availability depends on an interaction between PQLC2 and WDR41, the underlying mechanism for the regulated interaction is not known. In this study, we establish that the WDR41-PQLC2 interaction is mediated by a short peptide motif in a flexible loop that extends from the WDR41 β-propeller and inserts into a cavity presented by the inward-facing conformation of PQLC2. This data supports a transceptor model wherein conformational changes in PQLC2 related to substrate transport regulate the availability of the WDR41 binding site on PQLC2 and mediate recruitment of the WDR41-SMCR8-C9orf72 complex to the surface of lysosomes.

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Receptor-like role for PQLC2 amino acid transporter in the lysosomal sensing of cationic amino acids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014941118 ◽

2021 ◽

Vol 118 (8) ◽

pp. e2014941118 ◽

Cited By ~ 1

Author(s):

Gabriel Talaia ◽

Joseph Amick ◽

Shawn M. Ferguson

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Protein Complex ◽

Underlying Mechanism ◽

Amino Acid Transporter ◽

Cationic Amino Acid ◽

Flexible Loop ◽

Amino Acid Availability ◽

Cationic Amino Acids ◽

Acid Transporter

PQLC2, a lysosomal cationic amino acid transporter, also serves as a sensor that responds to scarcity of its substrates by recruiting a protein complex composed of C9orf72, SMCR8, and WDR41 to the surface of lysosomes. This protein complex controls multiple aspects of lysosome function. Although it is known that this response to changes in cationic amino acid availability depends on an interaction between PQLC2 and WDR41, the underlying mechanism for the regulated interaction is not known. In this study, we present evidence that the WDR41–PQLC2 interaction is mediated by a short peptide motif in a flexible loop that extends from the WDR41 β-propeller and inserts into a cavity presented by the inward-facing conformation of PQLC2. The data support a transceptor model wherein conformational changes in PQLC2 related to substrate transport regulate the availability of the WDR41-binding site on PQLC2 and mediate recruitment of the WDR41-SMCR8-C9orf72 complex to the surface of lysosomes.

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Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9

Molecular and Cellular Biology ◽

10.1128/mcb.00125-15 ◽

2015 ◽

Vol 35 (14) ◽

pp. 2479-2494 ◽

Cited By ~ 128

Author(s):

Jennifer Jung ◽

Heide Marika Genau ◽

Christian Behrends

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Transmembrane Protein ◽

Nutrient Status ◽

Protein Translation ◽

Dependent Manner ◽

Rag Gtpases ◽

Amino Acid Availability ◽

State Dependent ◽

Mtorc1 Activation

The serine/threonine kinase mTORC1 regulates cellular homeostasis in response to many cues, such as nutrient status and energy level. Amino acids induce mTORC1 activation on lysosomes via the small Rag GTPases and the Ragulator complex, thereby controlling protein translation and cell growth. Here, we identify the human 11-pass transmembrane protein SLC38A9 as a novel component of the Rag-Ragulator complex. SLC38A9 localizes with Rag-Ragulator complex components on lysosomes and associates with Rag GTPases in an amino acid-sensitive and nucleotide binding state-dependent manner. Depletion of SLC38A9 inhibits mTORC1 activity in the presence of amino acids and in response to amino acid replenishment following starvation. Conversely, SLC38A9 overexpression causes RHEB (Ras homolog enriched in brain) GTPase-dependent hyperactivation of mTORC1 and partly sustains mTORC1 activity upon amino acid deprivation. Intriguingly, during amino acid starvation mTOR is retained at the lysosome upon SLC38A9 depletion but fails to be activated. Together, the findings of our study reveal SLC38A9 as a Rag-Ragulator complex member transducing amino acid availability to mTORC1 activity.

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Early signalling events of autophagy

Essays in Biochemistry ◽

10.1042/bse0550001 ◽

2013 ◽

Vol 55 ◽

pp. 1-15 ◽

Cited By ~ 16

Author(s):

Laura E. Gallagher ◽

Edmond Y.W. Chan

Keyword(s):

Protein Kinase ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Mammalian Cells ◽

Mammalian Target Of Rapamycin ◽

Interacting Protein ◽

The Core ◽

Autophagy Gene ◽

Autophagy Induction ◽

Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

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