Changes in the cellular environment can lead to the reconfiguration of the landscape of plasma membrane proteins such as nutrient transporters. This occurs through their endocytosis, a selective process involving transporters conjugation to ubiquitin (Ub). Little is known concerning how nutrient fluctuations control transporter endocytosis, despite their essential role in cellular homeostasis. We aim at characterizing the mechanisms controlling these events in the case of nutrient starvation.
Task 1. Identification and characterization of cargoes regulated by glucose starvation.<br />a. What is the extent of starvation-induced endocytosis? <br />To use a combination of proteomics and imaging-based screen to determine the extent of the membrane protein rearrangement during glucose starvation in wild-type cells.<br />b. Which ARTs regulate which cargoes during starvation-induced endocytosis? <br />To study the involvement of the ART protein Csr2 in endocytosis during glucose starvation. <br /><br />Task 2. Molecular mechanisms of nutrient-starvation induced endocytosis<br />a. How do ART-Rsp5 complexes become activated upon starvation? <br />To study how changes in arrestin expression, post-translational modifications (phosphorylation and ubiquitylation) on its activity during glucose starvation, focusing on the ART protein Csr2. We will also characterize the signaling pathways involved.<br />b. How are cargoes recognized by ARTs in specific physiological conditions? <br />To obtain evidence of an interaction between Csr2 and its cargoes, and understand how it is regulated. <br /><br />Task 3 / Additional actors required for endocytosis during glucose starvation<br />We will exploit the results of a high-content imaging screen for mutants affected in endocytosis during glucose deprivation using an automated platform. We will prioritize candidates using information available in databases and select those with links to trafficking or nutrient-signaling pathways. We will check their involvement in starvation-induced endocytosis of GFP-tagged transporters (Task 1) and confirm the hits by various means
A combination of genetic screens, live cell imaging, transcription and proteomic analyses have revealed how yeast cells become operational for nutrient-starvation induced endocytosis. Strains expressing various GFP-tagged transporters were screened for their ability to endocytose the tagged transporters during nutrient starvation. Various mutants were then tested for their ability to perform this endocytosis. Once identified, the proteins in charge of this process were studied to understand their regulation in the physiological context of starvation. Analyses of their transcriptional and post-translational modifications revealed the molecular basis of this regulation. In particular, an innovative approach to study protein ubiquitylation based on split-luciferase complementation was developed in the partner lab (Rabut lab, IGDR/Rennes) and applied to our proteins of interest. Through genetic analyses, this was linked to nutrient-signaling pathways to integrate this in a broader context.
A combination of genetic screens, live cell imaging, transcription and proteomic analyses have revealed how yeast cells become operational for nutrient-starvation induced endocytosis. Our preliminary results suggested that glucose starvation induced the endocytosis of numerous transporters. A number of strains expressing various GFP-tagged transporters were screened for their ability to endocytose the tagged transporters during nutrient starvation. Various mutants were then tested for the inability to perform this endocytosis. Once identified, the proteins in charge of this process were studied to understand their regulation in the physiological context of starvation. Analyses of their transcriptional and post-translational modifications revealed the molecular basis of this regulation. This endocytosis differs from the endocytosis observed in glucose-replete cells, as it requires distinct ubiquitin ligase complexes regulated by novel mechanisms. In particular, an innovative approach to study protein ubiquitylation based on split-luciferase complementation was developed in the partner lab (Rabut lab IGDR/Rennes) and applied to our proteins of interest. Through genetic analyses, this regulation was linked to nutrient-signaling pathways to integrate this in a broader context. Parallel mechanisms were identified in the context of nitrogen starvation. We also investigated the regulation of endocytosis during a metabolic challenge by the glucose analogue 2-deoxyglucose.
Our results highlighted novel mechanisms by which cells regulate their surface proteins in response to nutrient challenges. This contributes to cell adaptation and viability during starvation and may relate to specific patho-physiological situations such as cancer.
Most of the objectives concerning the mechanistic understanding of the regulation of endocytosis during starvation (glucose or nitrogen) were reached and published. Some technical strategies did not succeed (eg. BioID, MS of membrane proteins) but were replaced by alternative strategies as detailed in the original project (eg. BiFC, imaging of transporters-GFP, respectively). Some aspects of the project such as the discovery of new regulators by high-throughput screening were complicated by the fact that many hits were not confirmed, leaving a few hits that were difficult to link to endocytosis and, at this stage of the project could no longer be exploited in the imparted time. However, novel and unexpected avenues of research were developed, such as the use of the metabolic inhibitor 2-deoxyglucose to mimic starvation and trigger endocytosis, now pursued in the lab through other fundings.
- Ivashov V, Zimmer J, Schwabl S, Kahlhofer J, Weys S, Gstir R, Jakschitz, T, Kremser, L, Bonn, G, Lindner, H, Huber, L, Léon, S, Schmidt, O, Teis, D (2020) Complementary a-arrestin-Rsp5 complexes control selective nutrient transporter endocytosis in response to amino acid availability. eLife 9:e58246
- Le Boulch M, Brossard A, Le Dez G, Léon S, Rabut G. (2020) Sensitive detection of protein ubiquitylation using a protein-fragment complementation assay. J Cell Sci 133: jcs243733
- Defenouillere Q#, Verraes A#, Laussel C, Friedrich A, Schacherer J, Léon S (2019) The regulation of HAD-like phosphatases by signaling pathways modulates cellular resistance to the metabolic inhibitor, 2-deoxyglucose. Science Signaling 12:eaaw8000
? Press release: insb.cnrs.fr/fr/cnrsinfo/une-strategie-de-resistance-un-inhibiteur-metabolique-decouverte-grace-la-levure
- Hovsepian J, Albanèse V, Becuwe M, Ivashov V, Teis D, Léon S (2018). The yeast arrestin-related protein Bul1 is a novel actor of glucose-induced endocytosis. Mol Biol Cell. 29:1012-1020.
- Hovsepian J, Defenouillère Q, Albanèse V, Váchová L, Garcia C, Palková Z, Léon S. (2017) Multilevel regulation of an a-arrestin by glucose depletion controls hexose transporter endocytosis. J Cell Biol. 216:1811-31.
? Press release: insb.cnrs.fr/fr/cnrsinfo/comment-les-cellules-sadaptent-elles-une-carence-en-sucre
The ability to respond to growth factors and nutrient fluctuations in the environment is essential for cell growth and survival. A key response to these changes in the environment is the reconfiguration of the protein composition in the plasma membrane. This involves the endocytosis of certain growth factor receptors and nutrient transporters from the plasma membrane. The endocytic removal of plasma membrane proteins is a highly selective process. It is mediated by their conjugation to ubiquitin (Ub), which acts as a molecular tag and drives their endocytosis. Once ubiquitylated, plasma membrane proteins undergo endocytosis and are sorted via the multivesicular body (MVB) pathway to the lumen of the lysosome where they are degraded. Thereby the ubiquitylation of plasma membrane proteins and their subsequent endocytic downregulation determines how cells respond and adapt to extracellular cues. The molecular mechanisms that lead to the ubiquitylation of growth factor receptors (e.g. the epidermal growth factor receptor), in response to ligand binding are well characterized and defects in this process contribute to pathologies such as cancer. In contrast, little is known how nutrient fluctuations control the ubiquitylation and endocytosis of nutrient transporters, despite their essential role in cellular homeostasis.
We have been among the first to provide insights into the molecular mechanisms responsible for the downregulation of nutrient transporters in response to nutrient excess in the medium (Léon laboratory; Becuwe et al., J Cell Biol 2012 and eLife 2014). Conversely, nutrient limitation induces selective endocytosis of many different plasma membrane proteins, including many nutrient transporters (Teis laboratory: Müller et al., eLife 2015). These findings revealed an essential function for starvation-induced endocytosis in cell survival. In this project we aim to provide a systems-level, mechanistic understanding into the molecular mechanisms driving the selective ubiquitylation and endocytosis of nutrient transporters under starvation conditions.
Our preliminary results suggest that glucose starvation induces the endocytosis of numerous transporters. This endocytosis differs in several aspects from the endocytosis observed in cells growing under nutrient replete conditions, as it requires distinct ubiquitin ligase complexes that are regulated by novel mechanisms. Now we wish to characterize in a systematic manner the extent to which glucose starvation regulates the plasma membrane proteome, and understand the underlying molecular events. So far, we identified at least two independent pathways that regulate endocytosis during glucose deprivation. We will study the impact of each of these pathways on plasma membrane remodeling during glucose deprivation, how these different regulatory systems select cargoes for endocytosis, and how they are regulated by nutritional inputs. This will provide novel insights into how this entire process contributes to the survival of starving cells.
To achieve these goals we will rely on the complementary expertise and numerous preliminary data from the Léon, Teis and Rabut laboratories and combine yeast genetics, imaging and quantitative proteomics. Our results will have broad biological implications and will help to understand how cells adjust their membrane proteome to specific growth conditions, which may be central to many metabolic diseases and cancer.
Monsieur Sébastien LEON (Institut Jacques Monod (IJM) - UMR7592 CNRS/Univ. Paris-Diderot)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IMU-Biocenter Innsbruck Medical University, Biocenter, Division Cell Biology
IGDR Institut de génétique et développement de Rennes (IGDR) - UMR6290 CNRS/Université Rennes 1
IJM Institut Jacques Monod (IJM) - UMR7592 CNRS/Univ. Paris-Diderot
Help of the ANR 268,012 euros
Beginning and duration of the scientific project: September 2016 - 36 Months