Fluorescence-mediated analysis of mitochondrial preprotein import in vitro
Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg, Germany.Analytical Biochemistry (Impact Factor: 2.22). 09/2006; 355(1):81-9. DOI: 10.1016/j.ab.2006.04.031
Mitochondrial biogenesis is a crucial element of the functional maintenance of a eukaryotic cell. The organelle must import the majority of its proteins from the cytosol where they are synthesized as precursors. In vitro import assays have been developed in which isolated mitochondria are incubated with precursor proteins, that are generated either by in vitro translation systems or by expression and purification as recombinant proteins. The detection of imported proteins is performed by autoradiography or by Western blot. We have now established a novel detection system for imported precursor proteins that is based on fluorescent labeling. We constructed a mitochondrial preprotein containing a C-terminal SNAP-tag that can label itself with a single fluorescein molecule in an enzymatic reaction. The fluorescent preproteins were efficiently imported into isolated mitochondria and showed kinetic behavior similar to that of standard preproteins. The fluorescence detection was sensitive and significantly faster than other comparable procedures. We also showed that precursor proteins containing a SNAP-tag domain could be successfully labeled in a postimport reaction in intact mitochondria. In summary, the use of a reporter domain modified with a fluorescent dye provides a novel, sensitive, and fast detection method to analyze the properties of the mitochondrial import reaction in vitro.
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ABSTRACT: A series of monoreactive functional benzo[a]phenoxazinium chlorides with a carboxyl, hydroxyl, amine or chloromethyl group were used as labels in the preparation of long-wavelength fluorescent amino acid bioconjugates. UV–visible and fluorescence studies of all compounds were carried out in ethanol and water at physiological pH. The absorption and emission of all compounds synthesised were in the range 555–640 nm and 632–681 nm, respectively.Graphical abstractTetrahedron 02/2007; 63(7):1654-1663. DOI:10.1016/j.tet.2006.12.005 · 2.64 Impact Factor
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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 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.Analytical Biochemistry 04/2007; 362(1):76-82. DOI:10.1016/j.ab.2006.12.015 · 2.22 Impact Factor
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ABSTRACT: The import of precursor proteins into mitochondria represents a cell biological process that is absolutely required for the survival of an eukaryotic cell. A complex chain of reactions needs to be followed to achieve a successful transport of mitochondrial proteins from the cytosol through the double membrane system to their final destination. In order to elucidate the details of the translocation process, in vitro import assays have been developed that are based on the incubation of isolated active mitochondria with natural or artificial precursor proteins containing the appropriate targeting information. Although most of the protein components of the import machinery have been identified and functionally characterized using this basic system, the definition of the molecular mechanisms requires more specialized assay techniques. Here we describe modifications of the standard in vitro import assay technique that are based on the utilization of recombinant preprotein constructs. The application of saturating amounts of substrate preproteins is a prerequisite for the determination of translocation kinetics and energy requirements of the import process. Accumulation of preproteins as membrane-spanning translocation intermediates further provides a basis for the functional and structural characterization of the active translocation machinery.Methods in Molecular Biology 02/2008; 457:59-83. DOI:10.1007/978-1-4939-2309-0_2 · 1.29 Impact Factor
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