Bates, George Thomas; (2023) Molecular mechanisms regulating CHC22 clathrin function. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Clathrins are self-assembling vesicle coat proteins that traffic molecules between membrane-bound compartments. Humans possess two isoforms of the clathrin heavy chain (CHC) subunits that share 85% sequence identity. CHC17 clathrin is ubiquitously expressed and plays a broad role in endocytosis and protein sorting. CHC22 clathrin is expressed primarily in muscle cells and functions to target the GLUT4 glucose transporter to an intracellular GLUT4 storage compartment (GSC). This compartment facilitates glucose uptake from the bloodstream by releasing GLUT4 to the cell surface in response to insulin, a key mechanism for glucose homeostasis. CHC22 sorts internalised GLUT4 from endosomes to the GSC, while newly synthesised GLUT4 relies on CHC22 for an unconventional Golgi-bypass route from the ER-to-Golgi intermediate compartment to the GSC. Both CHC isoforms must be recruited to distinct membrane sites to perform their distinct functions. This thesis research addresses how the molecular mechanism and regulation of CHC22 recruitment enables its functional distinction from CHC17. Using biochemistry and cell biology, a bipartite mechanism for membrane recruitment of CHC22 by the early secretory tether p115 was identified. This mechanism depends on both termini of CHC22. The N-terminal domain of CHC22 interacts directly with p115 through an isoform-specific patch absent in CHC17. In parallel, the C-terminal trimerisation domain (TxD) of CHC22 interacts indirectly with p115 via sorting nexin 5 (SNX5). Using x-ray crystallography, a high-resolution structure for the TxD was produced, showing key differences between CHC22 and CHC17, thereby providing insights into the potential molecular basis of the SNX5 interaction. These data lay the foundation for future investigation into the additional roles of SNX5 and TxD partners in CHC22 regulation and in understanding CHC22 endosome recruitment. As CHC22 plays a critical role in regulating GLUT4 traffic in human muscle, this molecular characterisation furthers understanding of human glucose metabolism and associated diseases.

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