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Carnitine palmitoyltransferase 1 (CPT1) controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for β-oxidation and has been reported to exist as an oligomer. We have investigated the in vivo oligomerization of full-length rat CPT1A (rCPT1A) along with those of the N-terminal truncation/deletion mutants Δ(1–82), Δ(1–18), and Δ(19–30) expressed in yeast mitochondria. The data indicate that in liver mitochondria in vivo CPT1A exists as a hexamer but that during preparation and storage of mitochondria the order of oligomerization is rapidly reduced to the trimer, such that a mixture of hexamer and trimer is observed in isolated mitochondria in vitro. Mutants bearing deletions of different segments of the N terminus (including the more N-terminal of the two transmembrane domains) have the same pattern of oligomerization when expressed in yeast mitochondria. The self-association of the individual rCPT1A transmembrane (TM) domains (TM1, TM2) was also studied using the TOXCAT assay (which measures TM self-association in the Escherichia coli inner membrane). There was minimal self-association of the sequence corresponding to TM1 but significant self-association of TM2 in TOXCAT. Chemical cross-linking and analytical ultracentrifugation of a TM2-derived synthetic peptide showed oligomerization with a similar trimer/hexamer equilibrium to that observed for native rCPT1A in isolated mitochondria. Therefore, there was a correlation between the oligomerization behavior of TM2 peptide and that of the full-length protein. In silico molecular modeling of rCPT1A TM2 highlighted the favorable orientation of GXXXG and GXXXA motifs in the formation of the TM2 hexamer.

Membrane proteins are involved in a wide range of vital cellular processes,\ud responsible for relaying signals and cargo across cell membranes. A deeper\ud molecular understanding of their function is essential to elucidate the mechanisms of\ud numerous diseases and medical conditions. As an example, the carnitine\ud palmitoyltransferase 1 (CPT1) enzymes are responsible for the regulation of\ud mitochondrial fatty acid oxidation thus are central to cell function.\ud Previous work had shown that the transmembrane (TM) domains of the mitochondrial\ud outer membrane protein CPT1 have a significant influence on the enzyme’s kinetics,\ud which is different in the two catalytically active isoforms, CPT1A and CPT1B. It was\ud also shown that TM-domains, and specifically TM2, are involved in driving the\ud oligomerisation of the full length CPT1A. For this reason, the study of TM-TM\ud oligomerisation and their roles in the function of CPT1 are fundamentally important in\ud the design of pharmacological strategies aimed at the modulation of the activities of\ud these enzymes in conditions such as diabetes.\ud The main focus of this PhD research was to systematically investigate the homo- and\ud hetero-oligomerisation of the TM domains to help better understand the structurefunction\ud relationship for CPT1 membrane proteins. In this project, different\ud techniques were used from the areas of chemistry, molecular biology, and biophysics\ud (in vivo oligomerisation assays, chemical crosslinking, circular dichroism and\ud analytical ultracentrifugation), in order to examine these interactions.\ud The membrane spanning sequences of rCPT1A and rCPT1B were found to selfassociate,\ud and the order of oligomerisation was also determined. Sequence motifs\ud likely to be responsible for the interactions of these TM domains were identified using\ud in silico modelling. Mutagenesis analyses confirmed the role of suggested GxxxG(A)\ud motifs in the homo-oligomerisation of rCPT1A TM2 domains. The heterooligomerisation\ud of TM1-TM2 domains was also studied, and was found to be\ud significant in both isoforms. The work presented in this thesis shows that the\ud membrane spanning regions of the CPT1 enzymes are capable of interacting through\ud both homo- and hetero-oligomerisation. The results strongly suggest that these\ud interactions may play a significant role in the complex formation of the full length\ud enzymes and provide further evidence that CPT1 may function as a channel in the\ud outer mitochondrial membrane.