Fluorescence-mediated analysis of mitochondrial preprotein import in vitro

ArticleinAnalytical Biochemistry 355(1):81-9 · September 2006with15 Reads
DOI: 10.1016/j.ab.2006.04.031 · Source: PubMed
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.
    • "In contrast to labeling strategies based on reactive amino groups or cysteines, this reaction generates labeling at a defined site and with 1 : 1 stoichiometry. A large variety of common reagents used in biology and biotechnology applications are already available, and both proteins and antibodies have been successfully covalently conjugated with fluorochromes, PEG, biotin, drugs, and other suitable molecules1234567891011121314. However, despite its reliability, the method has been designed for exclusive single tagging and is not suitable for applications, such as correlative microscopy, in which double labeling is mandatory. "
    [Show abstract] [Hide abstract] ABSTRACT: Antibodies are indispensable reagents in basic research, and those raised against tags constitute a useful tool for the evaluation of the biochemistry and biology of novel proteins. In this paper, we describe the isolation and characterization of a single-domain recombinant antibody (VHH) specific for the SNAP-tag, using Twist2 as a test-protein. The antibody was efficient in western blot, immunoprecipitation, immunopurification, and immunofluorescence. The sequence corresponding to the anti-SNAP has been subcloned for large-scale expression in vectors that allow its fusion to either a 6xHis-tag or the Fc domain of rabbit IgG2 taking advantage of a new plasmid that was specifically designed for VHH antibodies. The two different fusion antibodies were compared in immunopurification and immunofluorescence experiments, and the recombinant protein SNAP-Twist2 was accurately identified by the anti-SNAP Fc-VHH construct in the nuclear/nucleolar subcellular compartment. Furthermore, such localization was confirmed by direct Twist2 identification by means of anti-Twisit2 VHH antibodies recovered after panning of the same naïve phage display library used to isolate the anti-SNAP binders. Our successful localization of Twist2 protein using the SNAP-tag-based approach and the anti-Twist2-specific recombinant single-domain antibodies opens new research possibilities in this field.
    Full-text · Article · Oct 2010
    • "Chemically synthesized peptides have been used to investigate mitochondrial import (Pak and Weiner, 1990; Roise, 1992; Lu and Beavis, 1997 ), and have been conjugated to molecules , including small single-and double-stranded DNA (Vestweber and Schatz, 1989; Seibel et al., 1995 ), peptide nucleic acid (PNA)-coupled oligonucleotides (Flierl et al., 2003), liposome-encapsulated oligonucleotides (Geromel et al., 2001), and quantum dots (Hoshino et al., 2004 ). Mitochondrially targeted expression reporter plasmids have also been developed to investigate the mechanisms of mitochondrial import (Mukhopadhyay et al., 2003; Krayl et al., 2006), and nonmitochondrial proteins have been successfully delivered in a range of fusion proteins, including green fluorescent protein (GFP) (Kanazawa et al., 1997; Yano et al., 1997; Ni et al., 1999; Del Gaizo and Payne, 2003) and the cytosolic enzyme dihydrofolate reductase (DHFR) (Horwich et al., 1985a). The use of MTSs for the delivery of therapeutic molecules as both " synthetic " peptides and within fusion protein expression vectors is discussed in detail in the following sections . "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondrial DNA (mtDNA) disorders include a vast range of pathological conditions, despite each sharing a mutual inability to produce ATP efficiently as a result of defective oxidative phosphorylation. There is no clear consensus regarding an effective therapeutic approach, and consequently the current treatment strategies are largely supportive rather than curative. This is almost certainly the result of there being virtually no defined genotype-phenotype relationships among the mtDNA disorders; hence an identical mutation may be responsible for multiple phenotypes, or the same phenotype may be produced by different mutations. In light of this, the development of gene therapy to treat mtDNA disorders offers a promising approach, as it potentially circumvents the complication of the aforementioned genotype-phenotype inconsistency and ultimately the current inability to treat individual disorders with sufficient efficacy. Such an approach will ultimately require the combination of efficient mitochondrial targeting, and an effective therapeutic molecule. Although promising proof-of-principle developments in this field have been demonstrated, the realization of a successful therapeutic mitochondrial gene therapy strategy has not come to fruition. This review critiques the key approaches under development by discussing the theory underlying each strategy, and detailing the current progress made. We also emphasize the potential hurdles that must be acknowledged and overcome if the potential of a therapeutic gene therapy to treat mitochondrial DNA disorders is to be realized.
    Full-text · Article · Oct 2008
    • "This assay was validated using two mutants known to be differentially deficient in preprotein and presequence import. This assay has some similarities with the approach recently applied to preproteins [20], but does not require electrophoresis. Together, these assays could provide rapid screening of peptide and protein import mutants. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Apr 2007
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