RESUMEN
Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons. Here we show that phosphorylation of the ER-resident chaperone of nicotinic acetylcholine receptors, RIC-3, leads to increased muscle excitability. RIC-3 phosphorylation at Ser-164 depends on opposing functions of the phosphatase calcineurin (TAX-6), and of the casein kinase II homologue KIN-10. Effects of calcineurin down-regulation and of phosphorylated RIC-3 on muscle excitability are mediated by GABAA receptor inhibition. Thus RIC-3 phosphorylation enables effects of this chaperone on GABAA receptors in addition to nAChRs. This dual effect provides coordinated regulation of excitation and inhibition and enables fine-tuning of the excitation-inhibition balance. Moreover, regulation of inhibitory GABAA signaling by calcineurin, a calcium- and calmodulin-dependent phosphatase, enables homeostatic balancing of excitation and inhibition.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Calcineurina/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Quinasa de la Caseína II/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Chaperonas Moleculares/metabolismo , Neuronas Motoras/metabolismo , Músculos/metabolismo , Fosforilación , Receptores Nicotínicos/metabolismoRESUMEN
RIC-3 belongs to a conserved family of proteins influencing maturation of nicotinic acetylcholine receptors (nAChRs). RIC-3 homologues were shown to differently affect different nAChRs. Here we show that coexpression with RIC-3 increases the level of surface expression of DEG-3 while slightly reducing the level of surface expression of DES-2, both subunits of the DEG-3/DES-2 nAChRs. Those different effects are a likely explanation for the previously demonstrated effects of RIC-3, an endoplasmic reticulum resident protein, on properties of this receptor. To understand how RIC-3 interacts with different nAChR subunits, we identified and characterized domains and residues enabling this interaction. This analysis shows that conserved residues in the second RIC-3 transmembrane domain are needed for its interactions with two different Caenorhabditis elegans nAChRs, DEG-3/DES-2 and ACR-16. These conserved residues do not, however, function alone; rather, we show that additional domains also enable RIC-3's interactions with these receptors. Interestingly, the relative importance of these residues or of other domains mediating interactions of RIC-3 with nAChRs differs for the two different receptors. Differences in the way that RIC-3, predicted to be an intrinsically disordered protein, interacts with different receptors and receptor subunits suggest that it may adopt different conformations to enable these interactions. Such differences may explain both the effects of RIC-3 on receptor properties and the differences in its effects on different receptors.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Datos de Secuencia Molecular , Oocitos/metabolismo , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Receptores Nicotínicos/genética , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Fumarase and aconitase in yeast are dual localized to the cytosol and mitochondria by a similar targeting mechanism. These two tricarboxylic acid cycle enzymes are single translation products that are targeted to and processed by mitochondrial processing peptidase in mitochondria prior to distribution. The mechanism includes reverse translocation of a subset of processed molecules back into the cytosol. Here, we show that either depletion or overexpression of Cit2 (cytosolic citrate synthase) causes the vast majority of fumarase to be fully imported into mitochondria with a tiny amount or no fumarase in the cytosol. Normal dual distribution of fumarase (similar amounts in the cytosol and mitochondria) depends on an enzymatically active Cit2. Glyoxylate shunt deletion mutations (Deltamls1, Deltaaco1 and Deltaicl1) exhibit an altered fumarase dual distribution (like in Deltacit2). Finally, when succinic acid, a product of the glyoxylate shunt, is added to the growth medium, fumarase dual distribution is altered such that there are lower levels of fumarase in the cytosol. This study suggests that the cytosolic localization of a distributed mitochondrial protein is governed by intracellular metabolite cues. Specifically, we suggest that metabolites of the glyoxylate shunt act as 'nanosensors' for fumarase subcellular targeting and distribution. The possible mechanisms involved are discussed.