Molecular architecture of native LAMTOR and BORC assemblies

Lysosomes are intracellular signaling platforms that govern cell growth, division and differentiation. Despite great progress in our understanding of the molecular mechanisms of sensing such important nutrients as amino acids, cholesterol and glucose, key questions on how components of sensing and signaling machineries work together remain pending. The LAMTOR/Ragulator complex is a key regulator of lysosomal mTORC1, MAPK and AMPK signaling. LAMTOR also coordinates lysosome biogenesis via the BORC complex. We hypothesize that the LAMTOR complex exists in several functionally different states. The presence of distinct LAMTOR complexes on lysosomes raises fundamental questions on how anabolic and catabolic signaling, lysosomal biogenesis and positioning are coordinated on the mechanistic level to coordinate cellular fitness. We aim to identify all LAMTOR and BORC associated protein assemblies on intact lysosomes under different growth factors and nutrition conditions and obtain evidence for direct and endogenous protein-protein interactions. Thereby, we will determine the subunit topology of large protein assemblies. We will apply cross-linking mass spectrometry in combination with sophisticated subcellular fractionation to understand the molecular architectures of the different LAMTOR and BORC assemblies at endogenous levels and under different physiological conditions. A unique advantage of this approach is that native endogenous protein interactions are captured using a chemical cross-linker reactive towards specific amino acid side chains that are in close spatial proximity. We will apply sensitive technology and tools for the analysis of native LAMTOR and BORC protein complexes in knockout cells with reconstituted proteins to endogenous levels. Finally, endogenously tagged knockins will be generated. Both cell systems will be instrumental in detecting structural rearrangements in protein assemblies upon different physiological conditions. In a parallel approach, enriched lysosomes will be first cross-linked followed by affinity purification of protein complexes to reveal signaling-dependent rearrangements. Molecular mechanisms of how LAMTOR relates to different sensing and signaling protein machines, including BORC in particular, and how these interactions coordinate lysosomal biogenesis and function, to maintain cellular homeostasis, are largely unknown and are thus the focus of our interest.