SHY1 codes for a mitochondrial protein required for full expression of cytochrome oxidase (COX) in Saccharomyces cerevisiae. Mutations in the homologous human gene (SURF1) have been reported to cause Leigh's syndrome, a neurological disease associated with COX deficiency. The function of Shy1p/Surf1p is poorly understood. Here we have characterized revertants of shy1 null mutants carrying extragenic nuclear suppressor mutations. The steady-state levels of COX in the revertants is increased by a factor of 4-5, accounting for their ability to respire and grow on non-fermentable carbon sources at nearly wild-type rates. The suppressor mutations are in MSS51, a gene previously implicated in processing and translation of the COX1 transcript for subunit 1 (Cox1) of COX. The function of Shy1p and the mechanism of suppression of shy1 mutants were examined by comparing the rates of synthesis and turnover of the mitochondrial translation products in wild-type, mutant and revertant cells. We propose that Shy1p promotes the formation of an assembly intermediate in which Cox1 is one of the partners.
The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence. A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria. Mtg1p is a member of a family of GTPases with largely unknown functions. The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis. Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants. This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action. The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs. More likely, Mtg1p functions in assembly of the large ribosomal subunit. This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.
Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein.Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying noncompetitive antagonism at the nAChR.
The nicotinic acetylcholine receptor (nAChR)1 from the electric tissue of Torpedo californica is a prototype of the large family of ligand-gated ion channels (1, 2). It is an allosteric protein (3, 4) that can exist in at least three distinct, yet interconvertible conformational states (5): In the resting state the receptor has low affinity for acetylcholine and the ion channel is closed. Binding of two agonist molecules in a positively cooperative manner triggers gating of the intrinsic ion channel, which in turn leads to the permeation of cations. Prolonged agonist exposition induces receptor desensitization (6). In the desensitized conformation the nAChR has an increased affinity for acetylcholine as compared with the resting state, but the ion channel is closed. Each of these allosteric states is characterized by a distinct protein conformation.The structure of the nAChR meets basic prerequisites of allosteric regulatory proteins: First, it is an oligomer formed by five subunits with the stoichiometry ␣ 2 ␥␦. Second, given the homology of the subunits (7) and considering the fact that the neuronal ␣7-subunits can form functional homomeric receptors, one can assume that these subunits are arranged in a pseudo-symmetric manner. Indeed, in the pentamer the ␣-helical transmembrane segments M2 of each subunit contribute to a central ion channel (8), whereas the residual segments are oriented toward the plasma membrane or neighboring subunits. Third, the binding sites for agonists and competitive antagonists are located at the interfaces between neighboring subunits, i.e. in the case of Torpedo and of muscle-type nAChR an ␣-subunit and the adjacent ␥...
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