ABSTRACT
Rab GTPases and SNARE fusion proteins direct cargo trafficking through the exocytic and endocytic pathways of eukaryotic cells. We have used steady state mRNA expression profiling and computational hierarchical clustering methods to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators in 79 human and 61 mouse nonredundant tissues. We now show that this systems biology approach can be used to define building blocks for membrane trafficking based on Rab-centric protein activity hubs. These Rab-regulated hubs provide a framework for an integrated coding system, the membrome network, which regulates the dynamics of the specialized membrane architecture of differentiated cells. The distribution of Rab-regulated hubs illustrates a number of facets that guides the overall organization of subcellular compartments of cells and tissues through the activity of dynamic protein interaction networks. An interactive website for exploring datasets comprising components of the Rab-regulated hubs that define the membrome of different cell and organ systems in both human and mouse is available at http://www.membrome.org/.
Subject(s)
Gene Expression Profiling , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , Animals , Biological Transport , Humans , Internet , Lipid Metabolism , Mice , Multigene Family , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases/genetics , Phosphotransferases/metabolismABSTRACT
Prenylation of Rab GTPases regulating vesicle traffic by Rab geranylgeranyltransferase (RabGGTase) requires a complex formed by the association of newly synthesized Rab proteins with Rab-escort-protein (REP), the choroideremia-gene-product that is mutated in disease, leading to loss of vision. After delivery to the membrane by the REP-Rab complex, subsequent recycling to the cytosol requires the REP-related guanine-nucleotide-dissociation-inhibitor (GDI). Although REP and GDI share common Rab-binding properties, GDI cannot assist in Rab prenylation and REP cannot retrieve Rab proteins from the membranes. We have now isolated REP mutant proteins that are able to partially function as both REP and GDI. These results provide molecular insight into the functional and evolutionary organization of the REP/GDI superfamily.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Guanine Nucleotide Dissociation Inhibitors/metabolism , Saccharomyces cerevisiae/enzymology , rab GTP-Binding Proteins/metabolism , Alkyl and Aryl Transferases/genetics , Escherichia coli/genetics , Evolution, Molecular , Guanine Nucleotide Dissociation Inhibitors/genetics , Models, Structural , Mutation , Protein Binding , Protein Prenylation/physiology , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Signal Transduction , rab GTP-Binding Proteins/geneticsABSTRACT
Rab GTPases, key regulators of membrane targeting and fusion, require the covalent attachment of geranylgeranyl lipids to their C terminus for function. To elucidate the role of lipid in Rab recycling, we have determined the crystal structure of Rab guanine nucleotide dissociation inhibitor (alphaGDI) in complex with a geranylgeranyl (GG) ligand (H(2)N-Cys-(S-GG)-OMe). The lipid is bound beneath the Rab binding platform in a shallow hydrophobic groove. Mutation of the binding pocket in the brain-specific alphaGDI leads to mental retardation. Strikingly, lipid binding acts through a conserved allosteric switching mechanism to promote release of the GDI-Rab[GDP] complex from the membrane.
