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

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.

scholarly journals LARP1 facilitates translational recovery after amino acid refeeding by preserving long poly(A)-tailed TOP mRNAs

2019 ◽  
Author(s):  
Koichi Ogami ◽  
Yuka Oishi ◽  
Takuto Nogimori ◽  
Kentaro Sakamoto ◽  
Shin-ichi Hoshino
Keyword(s):  
Amino Acid ◽  
Ribosomal Proteins ◽  
Rna Binding ◽  
Tail Length ◽  
Underlying Mechanism ◽  
Growth Conditions ◽  
Mtor Inhibition ◽  
Terminal Oligopyrimidine ◽  
Related Proteins

ABSTRACTOccasionally, cells must adapt to an inimical growth conditions like amino acid starvation (AAS) by downregulating protein synthesis. A class of transcripts containing 5’terminal oligopyrimidine (5’TOP) motif encodes translation-related proteins such as ribosomal proteins (RPs) and elongation factors, and therefore, their translation is severely repressed during AAS to conserve energy1. The RNA-binding protein LARP1 transduces amino acid signaling to TOP gene expression by controlling translation and stability of TOP mRNAs2-6. When released from AAS, translation machineries in turn have to be restored, however, the underlying mechanism of such re-adaptation is largely unknown. Here we show that LARP1 preserves TOP mRNAs in a long polyadenylated state during long-term AAS. We found that TOP mRNAs become highly polyadenylated when cells are in AAS or treated with the mTOR (mechanistic target of rapamycin) inhibitor Torin1. Importantly, depletion of LARP1 completely abrogated the polyadenylation of TOP mRNAs. Comprehensive analysis of poly(A) tail length using the Nanopore direct RNA sequencing revealed that TOP mRNAs are selectively polyadenylated under mTOR inhibition. Since a long poly(A) tail confers increased stability and polysome formation of TOP mRNAs, we predict that LARP1-dependent preservation of TOP mRNAs enables rapid translational resumption after the release from AAS.

scholarly journals Positive epistasis between disease-causing missense mutations and silent polymorphism with effect on mRNA translation velocity

2021 ◽  
Vol 118 (4) ◽  
pp. e2010612118
Author(s):  
Robert Rauscher ◽  
Giovana B. Bampi ◽  
Marta Guevara-Ferrer ◽  
Leonardo A. Santos ◽  
Disha Joshi ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Amino Acid ◽  
Time Window ◽  
Mrna Translation ◽  
Underlying Mechanism ◽  
Missense Mutations ◽  
Translation Speed ◽  
Functional Studies ◽  
Translation Velocity ◽  
And Function

Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)—the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in cis exhibits positive epistatic effects on some CF disease–causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain–domain interactions that are otherwise perturbed by each individual mutation.

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

Biomolecules ◽  
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.

scholarly journals WDR41 supports lysosomal response to changes in amino acid availability

2018 ◽  
Vol 29 (18) ◽  
pp. 2213-2227 ◽  
Author(s):  
Joseph Amick ◽  
Arun Kumar Tharkeshwar ◽  
Catherine Amaya, ◽  
Shawn M. Ferguson
Keyword(s):  
Amino Acid ◽  
Protein Complex ◽  
Essential Role ◽  
Interacting Protein ◽  
Amino Acid Availability ◽  
Autophagy Induction ◽  
Mtorc1 Signaling ◽  
Parallel Pattern ◽  
Mtorc1 Activation ◽  
Lateral Sclerosis

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.

scholarly journals PtdIns3P controls mTORC1 signaling through lysosomal positioning

2017 ◽  
Vol 216 (12) ◽  
pp. 4217-4233 ◽  
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.

scholarly journals Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state

2018 ◽  
Author(s):  
Hsiang-Ting Lei ◽  
Jinming Ma ◽  
Silvia Sanchez Martinez ◽  
Tamir Gonen
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Signaling Pathway ◽  
Transmembrane Helix ◽  
Underlying Mechanism ◽  
Transitional State ◽  
Mechanistic Target Of Rapamycin ◽  
Mtorc1 Signaling ◽  
Solute Carrier ◽  
Amino Acid Sensing

Amino acid-dependent activation of mechanistic target of rapamycin complex 1 (mTORC1) is essential to reflect nutrient availabilities in cells for cell growth and metabolism1. Solute carrier 38 family A member 9 (SLC38A9) is the lysosomal transporter responsible for amino acid sensing in the mTORC1 signaling pathway2–4. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by the transmembrane helix 1 (TM1) of SLC38A9 which was anchored at the grove between transmembrane helix 5 and 7 inside the transporter. The key motif WNTMM on TM1, contributing to the anchoring interactions, is highly conserved in various species. Mutations in WNTMM motif abolished arginine transport by SLC38A9. The underlying mechanism of substrate binding is critical for both sensitizing mTORC1 signaling pathway to amino acids and for maintaining amino acid homeostasis across lysosomal membranes2.

Genetic dissection of Ragulator structure and function in amino acid-dependent regulation of mTORC1

2020 ◽  
Vol 168 (6) ◽  
pp. 621-632
Author(s):  
Shigeyuki Nada ◽  
Masato Okada
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Feedback Regulation ◽  
Genetic Dissection ◽  
Independent Manner ◽  
Negative Feedback Regulation ◽  
Rag Gtpases ◽  
And Function ◽  
Mtorc1 Activation ◽  
Membrane Scaffold

Abstract Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mechanistic target of rapamycin complex 1 (mTORC1). However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signalling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signalling.

scholarly journals Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

2021 ◽  
Vol 4 (3) ◽  
pp. 51
Author(s):  
Satish Kantipudi ◽  
Daniel Harder ◽  
Sara Bonetti ◽  
Dimitrios Fotiadis ◽  
Jean-Marc Jeckelmann
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Protein Complexes ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Expression Host ◽  
Amino Acids And Derivatives ◽  
Heterodimeric Complex ◽  
Acid Transporter ◽  
And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

Sign in / Sign up

Export Citation Format

Share Document