Structural & functional studies on cystinosin, the proton-coupled cystine transporter

Thesis

The lysosome is a major signalling centre within the cell and an important regulator of metabolism through its regulation of mTORC1 activity. A key function of the lysosome is to degrade proteins to their constituent amino acids and export these to the cytoplasm for metabolic recycling or sensing. In comparison to amino acid transport across the plasma membrane, transport across organellar membranes is currently poorly understood at both the cellular and molecular levels. Several physiological disorders are linked to mutations in lysosomal transporters, sparking a clear clinical and biochemical drive to understand their structure and mechanism of action. My thesis presents a series of structures of the lysosomal cystine transporter cystinosin (CTNS), captured in the ligand-bound inward-open and outward-open states. CTNS (SLC66A4), a member of the PQ-loop family, is responsible for exporting cystine from the lysosome using the pre-established proton gradient. Mutations in the CTNS gene lead to the accumulation of cystine in the lysosome, resulting in impaired lysosomal function, which leads to cystinosis, a rare disease that causes progressive glomerular damage and end-stage renal failure during mid-childhood. Until now, the molecular basis for proton-coupled cystine transport via CTNS remained elusive, hampering efforts to develop novel therapeutics for cystinosis and understand the role of CTNS in lysosomal homeostasis. The structural analysis of the transporter undertaken in this work reveals novel features in CTNS that are not observed in other PQ-loop proteins, such as the presence of two salt bridges that couple cystine transport to the translocation of protons. Using a combination of in vitro liposome assays targeting the Arabidopsis thaliana homologue of the transporter and in vivo experiments on human CTNS, I propose a mechanism for specific cystine recognition and proton-coupled transport. In contrast to current models regarding PQ-loop protein-mediated transport, the data indicate a more asymmetrical mode of action, exposing an interface which may allow CTNS to form functional dimers. Finally, the presented structures, coupled with the functional analysis of CTNS, provide a framework for understanding how specific hereditary defects cause cystinosis.

Authors: Löbel, M

• DOI: 10.5287/ora-o8r0yd2y0
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: lysosomal storage disease; cryo-EM; lysosomal transport; X-ray crystallography; nanobodies; cystinosin; cystinosis; transport mechanism; PQ-loop protein family
• Subjects: Biochemistry; Structural biology; Biophysics