guanosine-diphosphate and 2-bromopalmitate

The Rho family of GTPases comprises a major branch of the Ras superfamily of small GTPases. To date, at least 22 human members have been identified. However, most of our knowledge of Rho GTPase function comes from the study of the three classical Rho GTPases, RhoA, Rac1, and Cdc42. These Rho GTPases function as GDP/GTP-related binary switches that are activated by diverse extracellular signal-mediated stimuli. The activated GTPases then interact with downstream effectors to regulate cytoplasmic signaling networks that in turn regulate actin organization, cell cycle progression, and gene expression. Recently, studies have begun to explore the regulation and function of some of the lesser-known members of the Rho GTPase family. Wrch-1 (Wnt-regulated Cdc42 homolog-1) and the closely related Chp (Cdc42 homologous protein)/Wrch-2 protein comprise a distinct branch of the mammalian Rho GTPase family. Although both share significant sequence and functional similarities with Cdc42, Wrch proteins possess additional N- and C-terminal sequences that distinguish them from the classical Rho GTPases (Cdc42, RhoA, and Rac1). We have determined that Wrch-1 and Wrch2 exhibit unusual GDP/GTP binding properties and undergo posttranslational lipid modifications distinct from those of the classical Rho GTPases. In this chapter, we summarize our experimental approaches used to characterize the biochemical properties of these atypical Rho GTPases.

Topics: Animals; Cell Membrane; Chromatography, Affinity; Chromatography, Liquid; Escherichia coli; Glutathione Transferase; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Histidine; Humans; Mice; Neoplasm Proteins; NIH 3T3 Cells; Oligopeptides; Palmitates; Palmitic Acid; Recombinant Proteins; rho GTP-Binding Proteins

The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate. It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids. The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half-maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration. We conclude that the C-terminal region participates in the fatty acid regulation of UCP activity. The observed correlation between yeast growth rates in the presence of bromopalmitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cysteine; DNA Primers; Fatty Acids; Galactose; Guanosine Diphosphate; Ion Channels; Kinetics; Membrane Proteins; Mitochondrial Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxygen Consumption; Palmitates; Peptide Fragments; Permeability; Point Mutation; Recombinant Proteins; Saccharomyces cerevisiae; Uncoupling Protein 1