Abstract
Lysosome positioning is dynamically regulated by association with cytoskeletal motor proteins and direct contacts with other organelles. The intracellular position of lysosomes is intimately linked with their diverse functions, including their roles as the site of nutrient-dependent activation of the regulator of cell metabolism, mTORC1, and as the final degradative component of the endocytic and autophagic pathways. Lysosome distribution is therefore tightly regulated, although the molecular mechanisms involved are complex and poorlyunderstood.
The Folliculin (FLCN)- Folliculin-interacting protein (FNIP) complex is implicated in regulation of lysosome distribution and function. FLCN-FNIP regulates mTORC1 through GTPase-activating protein (GAP) activity towards lysosome-associated Rag C/D, in a manner dependent on its amino-terminal Longin domains and has been proposed to regulate lysosome positioning through interaction of its carboxy-terminal DENN domains with the Golgi
apparatus and lysosome associated proteins, Rab34 and RILP. The former is well characterised at a structural level, whilst less is understood about the latter.
This thesis explores how FLCN-FNIP interacts with RILP/Rab34 and interrogates the functional importance of this interaction. In Chapter 3 FLCN-FNIP complexes are expressed and purified with and without these binding partners. Initial characterisation of their molecular architecture is considered alongside a paralogous complex formed by SMCR8 and C9orf72. Chapter 4 combines electron cryo-microscopy with computational structure prediction,
crosslinking mass spectrometry and biochemical assays to define the binding site of RILP on the FLCN-FNIP complex and propose a new model for FLCN-FNIP-dependent autoregulation of RILP. Structural and biochemical findings are integrated with functional analysis in cells (Chapter 5), characterising the effects of disrupting the FLCN-FNIP-RILP-Rab complex through loss of FLCN and Rab34 on lysosome physiology and function.
This study establishes a ‘contact site model’ for starvation-induced perinuclear lysosome clustering mediated by tethering of lysosomes to the Golgi apparatus via the FLCN-FNIP2-RILP-Rab34 complex and provides greater mechanistic and functional understanding of the FLCN-related disorder Birt-Hogg-Dubé syndrome.
| Date of Award | 31 Jan 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Mark P Dodding (Supervisor) & Christiane H Berger-Schaffitzel (Supervisor) |