Assistant Professor of Biochemistry, Biophysics and Structural BiologyLab Homepage: http://www.robertozonculab.org/
How do the nutrients we consume regulate our growth and homeostasis? Answering this question will help us understand not only how we develop, but also how we age and why we become susceptible to diseases as diverse as cancer, diabetes and neurodegeneration. After decades of research, we know surprisingly little on how nutrients are sensed within cells, and how nutrient-derived signals remodel the cell and enable it to adjust to changing metabolic requirements. Many intracellular compartments, bearing exotic names such as mitochondria, lysosomes and autophagosomes, specialize in storing, releasing and processing metabolites that range from amino acids to sugars, lipids and nucleotides. But how does each organelle sense the quality and quantity of the metabolites it carries? And how is this nutrient and energy information of each organelle communicated to other compartments in the cell?
To tackle these questions, we focus on the lysosome as our model system. Using advanced live cell microscopy, in vitro biochemical assays, and high throughput protein and metabolite profiling, we are discovering wonderful new properties of this organelle, which has traditionally been viewed as the cell’s ‘trash can’. Instead, through our work the lysosome is emerging as a key signaling node, which relays nutrient availability to important signaling molecules such as the master growth regulator, mechanistic Target of Rapamycin Complex 1 (mTORC1) kinase (Figure 1).
The connection between the lysosome and mTORC1 has important implications for understanding the logic of metabolic regulation. mTORC1 drives biosynthetic processes such as ribosome biogenesis, mRNA translation and lipid synthesis. In turn, the lysosome mediates the breakdown of superfluous or damaged cellular components, thus providing a source of fuel and quality control for the cell. Our findings suggest that localized mTORC1 activation at the lysosome may provide a means for the cell to integrate biosynthetic and catabolic processes in space and time. Moreover, elucidating the connection between mTORC1 and the lysosome may point the way to novel approaches for pathologies in which mass accumulation and cellular quality control are deranged, primarily in cancer and protein misfolding diseases.
My postdoctoral work contributed to illuminating the mechanism through which amino acids activate mTORC1. We found that amino acids promote the translocation of mTORC1 from the cytoplasm to the lysosomal surface, where mTORC1 binds to a scaffolding complex composed of the Rag GTPases and Ragulator (Figure 2). Moreover, I found that amino acid signals from within the lysosomal lumen play an especially important role in mTORC1 activation. The proton pump, vacuolar H+ ATPase (V-ATPase), relays this ‘inside-out’ amino acid signal by binding to and regulating the Rag GTPases and Ragulator [Zoncu R. et al, Science (2011)].
Building on these findings, we will investigate the lysosome as a metabolic ‘command and control’ center that i) functions as a signaling hub for nutrient sensing and signaling and ii) controls the storage and delivery of key substrates to the cell’s metabolic pathways. Exploring these exciting directions poses significant challenges. We do not yet have a comprehensive list of the lysosomal resident proteins and internal metabolites. Moreover, we do not understand how the lysosome’s many parts work together to regulate biophysical properties of this organelle such as its internal acidity and membrane potential. We will meet these challenges by developing novel techniques to probe organelle function both in vivo and in vitro, and by integrating them with advanced live cell microscopy and high throughput approaches.
What are the mechanisms of nutrient sensing at the lysosome?
The ‘inside-out’ model of amino acid sensing poses intriguing questions: do amino acids bind to and modulate the V-ATPase from within the lysosomal lumen? How does the V-ATPase regulate the function of the mTORC1 scaffolding complex composed of Ragulator and Rag GTPases? Is the ATP hydrolysis rate and ADP/ATP ratio important? How does the proton gradient across the lysosomal membrane participate in amino acid sensing? We will address these questions using in vitro biochemical assays of mTORC1 binding and V-ATPase activity, in combination with live cell imaging experiments. Through collaborative work, we will gain structural insight into the overall assembly and regulation of this nutrient-sensing complex. These studies will shed light on how the lysosome senses its internal status and relays this information to downstream signaling effectors to regulate growth and metabolism.
How does the lysosome transport and store nutrients?
Our finding that the lysosome participates in amino acid sensing implies that this organelle must be able to dynamically exchange nutrients with other cellular compartments. To gain insight into how this is accomplished, we are developing techniques for the rapid and efficient capture of lysosomes from whole cells (Figure 3). Using time-resolved metabolite profiling of highly purified lysosomal preparations, we have obtained preliminary evidence that amino acids and other metabolites cross the lysosomal membrane bi-directionally in response to the nutritional status of the cell. However, we do not know the molecular identity of the transport systems of the lysosomal membrane, and how these transporters function together to regulate mTORC1 activity and cellular growth. Thus, we will combine proteomics-aided identification of candidate transporters with functional assays and in vitro reconstitution to shed light on the nutrient transport network of the lysosome. Finally, we will investigate how, in a class of diseases known as lysosomal storage disorders (LSDs), disruption of nutrient transport may contribute to metabolic derangement and cell death.
What regulates lysosomal composition and function?
Our work on amino acid sensing places the lysosome upstream of mTORC1. We recently found that, in turn, mTORC1 exerts a powerful regulatory action on lysosomal function and catabolism. Amino acids, acting via mTORC1, tightly control the activity of TFEB, a transcription factor that functions as a master regulator of lysosomal biogenesis and autophagy [Settembre C., Zoncu R. et al, EMBOJ (2012)] (Figure 4). This lysosome-to-nucleus signaling mechanism, which connects the lysosome’s internal status to catabolic gene expression programs, may be the ‘tip of the iceberg’ of a vast regulatory network for metabolic adaptation. To further explore this network, we will investigate how candidate mTORC1 and TFEB effectors, which we recently identified, control key properties of lysosomes, including their formation, trafficking and their ability to communicate and exchange nutrients with other important organelles.
In summary, our ultimate goals are to elucidate how organelles mediate nutrient sensing and utilization, and to illuminate the functional architecture of cellular metabolism in space and time.
Efeyan, A., Zoncu, R., Chang, S., Gumper, I., Snitkin, H., Wolfson, R., Oktay, K., Sabatini, D.D. and Sabatini, D.M. (2013) Rag GTPase-mediated regulation of mTORC1 by amino acids and glucose is necessary for neonatal autophagy and survival. Nature, 493, 679-83.
Efeyan, A.*, Zoncu, R.*¶, and Sabatini, D.M. ¶ (2012) Amino acids and mTORC1: from lysosomes to disease. Trends Mol Med, doi:10.1016/j.molmed.2012.05.007.
Bar-Peled, L., Schweitzer, L., Zoncu, R., and Sabatini, D.M. (2012) An expanded Ragulator is a GEF for the Rag GTPases that signal amino acid levels to mTORC1. Cell, 150, 1196-1208.
Settembre, C.*, Zoncu, R.*, Medina, D.L., Vetrini, F., Erdin, S., Erdin, S.U., Huynh, T., Ferron, M., Karsenty, G., Vellard, M.C., Facchinetti, V., Sabatini, D.M. and Ballabio, A. (2012) A lysosome-to-nucleus signaling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J. 31 (5): 1095-108.
Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y., and Sabatini, D.M. (2011) mTORC1 senses amino acids through a lysosomal inside-out mechanism that requires the Vacuolar H+-ATPase. Science, 334, 678-683.
Zoncu, R., and Sabatini, D.M. (2011) The TASCC of secretion. Science 332, 923-925.
Sheen, J.H., Zoncu, R., Kim, D., and Sabatini, D.M. (2011) Defective regulation of autophagy upon leucine deprivation reveals a targetable liability of human melanoma cells in vitro and in vivo. Cancer Cell 19, 613-628.
Zoncu, R.*, Efeyan, A.*, and Sabatini, D.M. (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12, 21-35
Sancak, Y.*, Bar-Peled, L.*, Zoncu, R., Markhard, A.L., Nada, S., and Sabatini, D.M. (2010) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290-303.
Zoncu, R.*, Perera, R.M.*, Balkin, D.M., Pirruccello, M., Toomre, D., and De Camilli, P. (2009). A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell 136, 1110-1121.
Zoncu, R., Perera, R.M., Sebastian, R., Nakatsu, F., Chen, H., Balla, T., Ayala, G., Toomre, D., and De Camilli, P.V. (2007). Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci U S A 104, 3793-3798.
* equal contributors ¶ corresponding author
Last Updated 2013-07-29