A fluorescence assay for peptide translocation into mitochondria.
Translocation of the presequence is an early event in import of preproteins across the mitochondrial inner membrane by the TIM23 complex. Import of signal peptides, whose sequences mimic mitochondrial import presequences, was measured using a novel, qualitative, fluorescence assay in about 1h. This peptide assay was used in conjunction with classical protein import analyses and electrophysiological approaches to examine the mechanisms underlying the functional effects of depleting two TIM23 complex components. Tim23p forms, at least in part, the pore of this complex while Tim44p forms part of the translocation motor. Depletion of Tim23p eliminates TIM23 channel activity, which interferes with both peptide and preprotein translocation. In contrast, depletion of Tim44p disrupts preprotein but not peptide translocation, which has no effect on TIM23 channel activity. Two conclusions were made. First, this fluorescence peptide assay was validated as two different mutants were accurately identified. Hence, this assay could provide a rapid means of screening mutants to identify those that fail an initial step in import, i.e., translocation of the presequence. Second, translocation of signal peptides required normal channel activity and disruption of the presequence translocase-associated motor complex did not modify TIM23 channel activity nor prevent presequence translocation.
Martinez-Caballero
Sonia
S
Department of Basic Sciences, College of Dentistry, New York University, New York, NY 10010, USA.
Peixoto
Pablo M V
PM
Kinnally
Kathleen W
KW
Campo
María Luisa
ML
D005456:Fluorescent Dyes / Q000737:chemistry*; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; C083081:TIM23 protein, S cerevisiae; C077016:TIM44 protein, S cerevisiae