title: Awaking TIM22, a dynamic ligand-gated channel for protein insertion in the mitochondrial inner membrane.
abstract: Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Extremadura, 10071 Cáceres, Spain.
authors.forename: Pablo M V
authors.initials: PM
authors.lastname: Peixoto
authors.forename: Fernando
authors.initials: F
authors.lastname: Graña
authors.forename: Teresa J
authors.initials: TJ
authors.lastname: Roy
authors.forename: Cory D
authors.initials: CD
authors.lastname: Dunn
authors.forename: Montaña
authors.initials: M
authors.lastname: Flores
authors.forename: Robert E
authors.initials: RE
authors.lastname: Jensen
authors.forename: María Luisa
authors.initials: ML
authors.lastname: Campo
mesh_terms: D015640:Ion Channel Gating / Q000502:physiology*; D008024:Ligands; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D033681:Mitochondrial Membrane Transport Proteins; D051336:Mitochondrial Membranes / Q000502:physiology*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D008024:Ligands; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; C102549:TIM22 protein, S cerevisiae; C083081:TIM23 protein, S cerevisiae