The identification and characterisation of Rab4 interacting proteins

Rab4 is a member of the Rab superfamily of small GTPases. It is localized to the early sorting endosome and plays a role in regulating the transport from this compartment to the recycling and degradative pathways. In order to further our understanding of the role Rab4 plays in endocytosis, a yeast two-hybrid screen was performed to identify putative Rab4 effectors. A constitutively active mutant of Rab4, Rab4Q67L, when used as bait to screen a HeLa cDNA library, identified a novel 80kDa protein that interacted with Rab4-GTP. This protein was called Rab Coupling Protein (RCP). RCP interacts preferentially with the GTP-bound form of Rab4. Subsequent work demonstrated that RCP also interacts with Rab11, and that this interaction is not nucleotide-depenedent. RCP is predominantly membrane-bound and localised to the perinuclear recycling compartment. Expression of a truncation mutant of RCP, that contains the Rab binding domain, in HeLa cells, results in the formation of an extensive tubular network that can be labelled with transferrin. These tubules are derived from the recycling compartment since they are inaccessible to transferrin when the ligand is internalised at 18oC. The truncation mutant-induced morphology can be rescued by overexpression of active Rab11, but not active Rab4. This suggests that RCP functions between Rab4 and Rab11 in the receptor recycling pathway, and may act as a ‘molecular bridge’ between these two sequentially acting small GTPases. Quantitative assays demonstrated that overexpression of the truncation mutant results in a dramatic inhibition in the rate of receptor recycling. Database analysis revealed that RCP belongs to a family of Rab interacting proteins, each characterised by a carboxy-terminal coiled-coil domain and an amino-terminal phospholipid-binding domain. KIAA0941, an RCP homologue, interacts with Rab11, but not with Rab4. Overexpression of its Rab binding domain also results in a tubular network, however, this tubulation cannot be rescued by active Rab11.

An investigation of the functional role of GRAB in the context of Rab3, Rab8 and Rab11

Rab GTPases are the largest family of the Ras superfamily and are key regulators of membrane trafficking within the cell. There are over 60 members of the Rab family which localise to specific membrane compartments and interact with effector proteins to regulate membrane trafficking processes, such as vesicle formation, vesicle trafficking within the cell and fusion with an acceptor compartment. Multiple effector proteins have been identified for many Rabs, some of which can interact with more than one Rab to link their function at a specific membrane location or to link them together in a Rab activation cascade. Rabin8 is one such protein which is an effector for Rab11a and a Guanine nucleotide Exchange Factor (GEF) for Rab8a. Rabin8 participates in a conserved Rab activation cascade which is critical in the formation of primary cilia. Data presented in this thesis has shown that GRAB interacts with Rab3a, Rab8a, Rab11a and Rab11b in a nucleotide dependent manner. Furthermore, the minimal interacting regionbetween these proteins has been investigated. The functional outcome of GRAB knockdown has also been examined and data in this thesis highlights the phenotypic outcome.