Structure-Function analysis of the late endosomal LAMTOR complex

Scaffold and adaptor proteins organize signaling protein complexes at specific subcellular locations and regulate signal transduction in different cellular processes such as proliferation, differentiation, apoptosis and migration. We have shown previously that the Mitogen-activated protein kinase (MAPK) scaffold protein 1 (MP1) is localized to late endosomes by the adaptor protein p14 (1). The p14 protein forms a stable heterodimeric complex with MP1 which binds MEK1, ERK1/2, recruits the entire complex to the late endosome via the p18 anchor protein, and thus facilitates signal transduction through the MAPK cascade on this specific subcellular location by providing critical spatial and temporal specificity (2,3). Recently, it was shown that the p18/p14/MP1 (LAMTOR) complex mediated the translocation of mammalian target of rapamycin complex 1 (mTORC1) to the lysosomal surface (4) and that the extended p18/p14/MP1/HBXIP/C7orf59 complex serves as a guanine nucleotide exchange factor (GEF) to RagA and RagB GTPases, thereby signaling amino acid levels to mTORC1(5). mTORC1 regulates cell growth by integrating upstream signals from growth factors as well as nutrient and intracellular energy levels. Using conditional gene disruption of p14 in mice we have previously demonstrated that the p14/MP1 complex regulates late endosomal traffic and cellular proliferation (6). Furthermore, we could show that a novel human immunodeficiency syndrome caused by genetic deficiency of the p14 leads to aberrant lysosomal function (7). In addition, the p18-mTORC1 pathway has been recently implicated in terminal maturation of lysosomes (8). Taken together, these data highlight the role of the endosomal scaffold complex p18/p14/MP1 not only as a convergence point of MAPK and mTORC1 signaling pathways but also as a key regulator of endosomal biogenesis. What is the novelty of the project proposed here? The proposed work will deliver a detailed structural analysis of the extended LAMTOR complex. The structural information will be complemented with functional assays to determine how the different biological functions are executed and coordinated. Our working hypothesis is that the LAMTOR complex assembles as a TRAPP (trafficking protein particle) like multimeric platform on late endosomes that coordinates signaling and late endosomal biogenesis. The principal questions to be answered by the following proposal are: (1) In which stoichiometry and variants does the LAMTOR complex assemble? (2) Is there a common principle/similarity to other biological complexes? (3) How does the LAMTOR complex regulate endosomal biogenesis? In order to answer these questions we aim to (1) Determine the structure of the pentameric LAMTOR complex, (2) perform a comparative analysis to other structurally similar complexes present in other intracellular membrane compartments, in particular the TRAPP complex, and (3) investigate if a putative SNARE binding domain is present in the LAMTOR complex. Taken together, the proposed studies are expected to add mechanistic insight into the role of the LAMTOR complex in the coordination of intracellular signaling and endosomal biogenesis.

Cellular homeostasis is intrinsically dependent on the coordinated function of lysosomal degradation and cellular metabolism. In fact, a defective interplay between the two processes, underlies many human pathologies, including neurodegeneration, cancer, infection, immunodeficiency and obesity. Over the years, our team has contributed towards the identification and functional characterization of a pentameric lysosomal protein complex named LAMTOR. This complex acts not only as a critical hub coordinating MAPK and mTORC signaling from the lysosomal membrane, but also as a key regulator of lysosomal biogenesis and intracellular positioning. Within the framework of this project, we performed a detailed structural analysis of LAMTOR and its association with the RagGTPases. The structural information was complemented with functional assays essential to determine how the different LAMTOR dependent functions are executed and coordinated. Pentameric LAMTOR, assembles in a Lego brick manner. Four of the LAMTOR subunits assume an almost identical structural fold and assemble as two individual roadblock/LC7 heterodimers wrapped and held together by the fifth component, LAMTOR1, which anchors the complex on lysosomes. In addition, the Rag GTPases associate with the LAMTOR complex through their C-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components. Though mTORC1 dysfunction is indisputably linked to many disease conditions such as cancer and inflammation/immunity, the use of Rapalogs and catalytic mTOR inhibitors has been hampered by only partial inhibition of substrates, activation of resistance and severe dose limiting toxicities. One interesting avenue to generate mTORC1 specific modulators is the targeting of upstream regulators. In this context, altering the Rag-LAMTOR interaction might represent a novel mechanism for specific inhibition of mTORC1. Within this project we identified and functionally validated two small structural motifs (LVV and NIV) in LAMTOR1, necessary for Rags recruitment. This knowledge may be used in future to design compounds interfering with this interaction and thereby being suitable as new medications in diseases where mTORC1 is dysregulated.