5] Antibody and oligonucleotide probes to distinguish intracellular expression and localization patterns of rab GDP-dissociation inhibitor isoforms

Abstract

This chapter describes a strategy to distinguish the endogenous expression and localization patterns of the guanosine diphosphate ( GDP) dissociation inhibitor (GDI) family members. Methods used to express and purify GDI-2 and GDI- β in bacterial hosts are also described. GDIs likely represent the only proteins fully competent to chaperone Rabs to their correct destination. Consistent with this requirement are several reports documenting additional important functions of GDI on membranes. Hence, GDI-1 contributes to the specific recognition of the Rab membrane targets, stimulates GTP/GDP exchange, and dissociates thereafter back into the cytosol, a step likely promoted by GDP dissociation factor(s) and cellular redox state. Three mammalian GDI genes, GDI-1, GDI-2, and GDI- β , have been cloned to date. They encode ubiquitously expressed, highly homologous protein isoforms, tightly conserved in mammals. The three GDIs are found equally capable of operating with various Rabs in cell-free systems, indicating that, at least in resting cells, no specificity toward an individual Rab or subset of Rab proteins is associated with GDI function. However, given the differential intracellular localization of GDI-1 vs. GDI-2 and the subtle regulation of Rab-GDI complex formation in the context of living cells, functional differences among GDIs can be anticipated.