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Cysteine residues are not essential for uncoupling protein function.

ABSTRACT: The uncoupling protein (UCP) of brown adipose tissue is a regulated proton carrier which allows uncoupling of mitochondrial respiration from ATP synthesis and, therefore, dissipation of metabolic energy as heat. In this article we demonstrate that, when UCP is expressed in Saccharomyces cerevisiae, it retains all its functional properties: proton and chloride transport, high-affinity binding of nucleotides and regulation of proton conductance by nucleotides and fatty acids. Site-directed mutagenesis demonstrates that sequential replacement by serine of cysteine residues in the UCP does not affect either its uncoupling activity or its regulation by nucleotides and fatty acids, and therefore establishes that none of the seven cysteine residues present in the wild-type UCP is critical for its activity. These data indicate that transport models involving essential thiol groups can be discounted and that chemical modification data require critical re-evaluation.

SUBMITTER: Arechaga I

PROVIDER: S-EPMC1137752 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

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Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

Project description:Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

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Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function.

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Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila.

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Allosteric Pathways Originating at Cysteine Residues in Regulators of G-Protein Signaling Proteins.

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| S-EPMC7895990 | biostudies-literature

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