RESUMEN

The yeast Saccharomyces cerevisiae genome is endowed with two distinct isoforms of Voltage-Dependent Anion Channel (VDAC). The isoform yVDAC2 is currently understudied with respect to the best known yVDAC1. Yet, since the discovery, the function of yVDAC2 was unclear, leading to the hypothesis that it might be devoid of a channel function. In this work we have elucidated, by bioinformatics modeling and electrophysiological analysis, the functional activity of yVDAC2. The conformation of yVDAC2 and, for comparison, of yVDAC1 were modeled using a multiple template approach involving mouse, human and zebrafish structures and both showed to arrange the sequences as the typical 19-stranded VDAC ß-barrel. Molecular dynamics simulations showed that yVDAC2, in comparison with yVDAC1, has a different number of permeation paths of potassium and chloride ions. yVDAC2 protein was over-expressed in the S. cerevisiae cells depleted of functional yVDAC1 (Δpor1 mutant) and, after purification, it was reconstituted in artificial membranes (planar lipid bilayer (PLB) system). The protein displayed channel-forming activity and the calculated conductance, voltage-dependence and ion selectivity values were similar to those of yVDAC1 and other members of VDAC family. This is the first time that yVDAC2 channel features are detected and characterized.

Asunto(s)

Mitocondrias/química , Membranas Mitocondriales/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/química , Canal Aniónico 2 Dependiente del Voltaje/química , Animales , Sitios de Unión , Cloruros/química , Cloruros/metabolismo , Biología Computacional , Expresión Génica , Humanos , Transporte Iónico , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Ratones , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Simulación de Dinámica Molecular , Potasio/química , Potasio/metabolismo , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología Estructural de Proteína , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/genética , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Pez Cebra