Pozo, Karine; (2007) Molecular studies of GRIF-1 and GRIF-1 interacting proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Communication between neurones in brain occurs at specialised zones, the synapses. The transport of proteins and organelles to these active regions relies on motor proteins. Motors associate with their cargoes either directly or via adaptor proteins. GABAA receptor interacting factor-1, GRIF-1, is thought to function as a scaffold linking kinesin-1 to mitochondria and/or vesicle-enclosed GABAA receptors for transportation to synapses. A confocal microscopy approach was used to investigate the association between GRIF-1 and the prototypic kinesin-1, KIF5C, GRIF-1 and the GABAA receptor and GRIF-1 and the enzyme, O-linked N-acetylglucosamine transferase (OGT). Enhanced yellow or cyan fluorescent (EYFP, ECFP respectively) fusion proteins of GRIF-1, KIF5C, the motor domain of KIF5C, the non-motor domain of KIF5C, the GABAA receptor subunit and OGT were generated. The fusion proteins were characterised and their distribution in mammalian cell lines was examined. To characterise further GRIF- 1/KIF5C interactions, fluorescence resonance energy transfer analyses were carried out. These studies showed that ECFP-GRIF-1 was associated directly with the carboxyl-terminal, non-motor domain of EYFP-KIF5C. Cellular organelles in cells overexpressing ECFP-GRIF-1 and EYFP-KIF5C were fluorescently labelled. In these cells, mitochondria were found re-distributed to GRIF-1 enriched regions. Searching for novel GRIF-1 associated proteins, a yeast two-hybrid cDNA library screen using the bait, GRIF-1 (545-913), was carried out and a partial cDNA encoding an interactor was isolated. Characterisation of this cDNA revealed that it encoded a mitochondrial isoform of OGT. The potential use of the CytoTrap yeast two-hybrid system to identify new GRIF-1 interactors was investigated. A full length GRIF-1 bait fusion protein was generated, characterised and the CytoTrap yeast two-hybrid system evaluated. To conclude, the analysis of GRIF-1 and its associated proteins indicates a role for GRIF-1 as an adaptor linking kinesin-1 to its cargoes, i.e. mitochondria and vesicle- enclosed GABAA receptors. This association may be regulated by the enzyme OGT.

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