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Characterisation of the involvement of rab5 in early endosome fusion

Steele-Mortimer, Olivia Ann; (1994) Characterisation of the involvement of rab5 in early endosome fusion. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The rab5 protein is a member of the rab family of ras-related GTPases, which are known to be involved in the regulation of membrane transport in eukaryotic cells. Rab5 is localised to early endosomes, clathrin coated vesicles and the plasma membrane and has been shown to regulate an early step in the endocytic pathway both in vivo and in vitro. Although the exact function of the rab proteins is still unknown, their activity is believed to be dependent on their ability to cycle between GTP- and GDP-bound forms. A cell-free assay has been used to characterise the role of rab5 in early endosome fusion. All three isoforms, rab5a, rab5b and rab5c, showed the same in vitro activity. The nucleotide requirement of rab5a was investigated using mutant proteins. The results of these experiments indicate that GTP-binding is required for rab5 activity, and that hydrolysis is required for inactivation and recycling of the protein. Data was also obtained indicating that the N-terminal domain of rab5 is required for its function. Two phosphoproteins were detected which specifically co-immunoprecipitate with rab5. One of these was identified as the regulatory protein Rab-GDI. Although a small fraction of GDI was found on membranes, phosphorylated GDI was detected only in cytosol fractions. It is proposed that phosphorylation/dephosphorylation of GDI can regulate the specificity and directionality of the rab protein cycle. Finally, the ability of the REP-1 protein to deliver rab proteins to membranes was investigated. Purified REP-1/rab5 complex significantly stimulated endosome fusion, confirming the hypothesis that REP-1 is an escort protein. This system should now enable us to study in detail the requirements for GDI and REP proteins in rab protein regulation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Characterisation of the involvement of rab5 in early endosome fusion
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Endosome fusion
URI: https://discovery.ucl.ac.uk/id/eprint/10107005
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