Fullwood, Yvonne; (2000) The cloning and characterisation of a novel mammalian phosphatidylinositol transfer protein, MrdgBβ. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phosphatidylinositol transfer proteins (PtdIns-TPs) have historically been described as proteins that constitutively catalyse the exchange of phospholipid monomers between membrane bilayers, thereby facilitating lipid distribution within cells. Recent data have identified an acute requirement for PtdIns-TPs during several essential cellular processes. The various PtdIns-TPs, including PITPα, PITPβ, retinal degeneration B (rdgB) and PYK2 amino-terminal domain interacting (Nir) proteins, can be divided into two structural families. The small, soluble PITP isoforms contain only a PtdIns transfer domain and have been implicated in phosphoinositide signalling and vesicle trafficking. In contrast, the rdgB proteins, which include the Nir proteins, contain an amino-terminal PITP-like domain, an acidic, Ca2+-binding domain, six putative transmembrane domains, and a conserved carboxy-terminal domain. Although the rdgB protein was originally identified as an invertebrate phototransduction protein, the biological function of rdgB proteins in vertebrates is unclear. This thesis describes the molecular cloning and characterisation of a novel rdgB protein, rdgBβ. The 38kDa, mammalian rdgBβ (MrdgBβ) protein contains an amino-terminal PITP-like domain and a short carboxy-terminal domain. Cytogenetic analysis reveals that the human rdgBβ gene is localised on chromosome 17q23. In contrast to other rdgB-like proteins, MrdgBβ contains neither transmembrane motifs nor the conserved carboxy-terminal domain. Northern analysis has demonstrated that MrdgBβ mRNA is ubiquitously expressed. Furthermore, immunofluorescence analysis of ectopic MrdgBβ showed cytoplasmic localisation. The phospholipid-binding specificity and transfer activities of MrdgBβ, relative to other PtdIns-TPs, have been addressed using radiolabelled- and pyrene-labelled phospholipid transfer assays. The functional significance of the results of these assays is discussed. While early reports found functional degeneracy in vitro, the work presented in this thesis supports more recent results, which suggest that different members of the PtdIns-TP family have distinct functions in vivo.

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