A fluorescence assay for peptide translocation into mitochondria

Sonia Martinez-Caballero, P. M V Peixoto, Kathleen W. Kinnally, María Luisa Campo

Research output: Contribution to journalArticle

Abstract

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 1 h. 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.

Original languageEnglish (US)
Pages (from-to)76-82
Number of pages7
JournalAnalytical Biochemistry
Volume362
Issue number1
DOIs
StatePublished - Mar 1 2007

Fingerprint

Mitochondria
Assays
Fluorescence
Peptides
Protein Sorting Signals
Mitochondrial Membranes
Screening
Membranes
Proteins

Keywords

  • Fluorescence assay
  • Mitochondria
  • Patch clamp
  • Protein import
  • Tim23
  • Tim44

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Martinez-Caballero, S., Peixoto, P. M. V., Kinnally, K. W., & Campo, M. L. (2007). A fluorescence assay for peptide translocation into mitochondria. Analytical Biochemistry, 362(1), 76-82. https://doi.org/10.1016/j.ab.2006.12.015

A fluorescence assay for peptide translocation into mitochondria. / Martinez-Caballero, Sonia; Peixoto, P. M V; Kinnally, Kathleen W.; Campo, María Luisa.

In: Analytical Biochemistry, Vol. 362, No. 1, 01.03.2007, p. 76-82.

Research output: Contribution to journalArticle

Martinez-Caballero, S, Peixoto, PMV, Kinnally, KW & Campo, ML 2007, 'A fluorescence assay for peptide translocation into mitochondria', Analytical Biochemistry, vol. 362, no. 1, pp. 76-82. https://doi.org/10.1016/j.ab.2006.12.015
Martinez-Caballero S, Peixoto PMV, Kinnally KW, Campo ML. A fluorescence assay for peptide translocation into mitochondria. Analytical Biochemistry. 2007 Mar 1;362(1):76-82. https://doi.org/10.1016/j.ab.2006.12.015
Martinez-Caballero, Sonia ; Peixoto, P. M V ; Kinnally, Kathleen W. ; Campo, María Luisa. / A fluorescence assay for peptide translocation into mitochondria. In: Analytical Biochemistry. 2007 ; Vol. 362, No. 1. pp. 76-82.
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