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First published online 24 June 2008
doi: 10.1242/jcs.029207

Journal of Cell Science 121, 2423-2431 (2008)
Published by The Company of Biologists 2008
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The mitochondrial targeting sequence tilts the balance between mitochondrial and cytosolic dual localization

Neta Regev-Rudzki*, Ohad Yogev* and Ophry Pines{ddagger}

Department of Molecular Biology, Hebrew University Medical School, Jerusalem 91120, Israel


Figure 1
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  		Fig. 1. MTS-exchange mutants. (A) Growth and complementation of aconitase and fumarase-MTS-exchange mutants. {Delta}aco1 or {Delta}fum1 strains harboring the indicated plasmids that encode MTS-exchange mutants were diluted and grown on galactose or ethanol-acetate medium plates as indicated (B) Aconitase- and fumarase-MTS-exchange mutants are processed. Wild-type, {Delta}aco1 and {Delta}fum1 strains induced for expression the indicated plasmids were labeled with [35S]methionine for 30 minutes, either in the absence (–) or presence (+) of 20 µM CCCP. Total cell extracts were prepared, immunoprecipitated with the indicated antiserum and analyzed using SDS-PAGE. Arrows show positions of precursor (p) and mature proteins (m). (C) MTS-exchange mutants exhibit alterations in the protein subcellular distribution. Yeast cells expressing aconitase and fumarase variants were subjected to subcellular fractionation. Equivalent portions from the total (Tot) cytosolic (Cyt) and mitochondrial (Mit) fractions were analyzed by western blotting using antibodies against the indicated proteins. Representative examples of mitochondrial (Hsp60) and cytosolic (hexokinase1, HK) controls are shown. Cytosolic and mitochondrial band intensities were quantified densitometrically using TINA Software.

 

Figure 2
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  		Fig. 2. Aconitase-MTS point mutations. (A) Growth and complementation of aconitase-MTS point mutants. {Delta}aco1 strain harboring the indicated plasmids encoding MTS point mutants were diluted and grown on galactose or ethanol-acetate medium plates as indicated. (B) Aconitase-MTS point mutants are processed. MTS point mutants of aconitase were labeled and examined for processing as described in the legend to Fig. 1B. (C) MTS point mutants exhibit alterations in the protein subcellular distribution. Yeast cells expressing aconitase variants were subjected to subcellular fractionation. Equivalent portions from the total (Tot) cytosolic (Cyt) and mitochondrial (Mit) fractions were analyzed by western blotting using antibodies against the indicated proteins.

 

Figure 3
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  		Fig. 3. Aconitase-MTS-elongated mutants. (A) Growth and complementation of aconitase-MTS elongated mutants. {Delta}aco1 strains harboring the indicated plasmids encoding MTS elongated mutants were diluted and grown on galactose or ethanol-acetate medium plates as indicated. (B) Aconitase elongated mutants are processed. MTS-elongated mutants of aconitase were labeled and examined for processing as described in the legend to Fig. 1B. (C) Aconitase elongated mutants exhibit alterations in the protein subcellular distribution. Subcellular fractionations of yeast cells expressing aconitase elongated mutants. Equivalent portions from the total (Tot) cytosolic (Cyt) and mitochondrial (Mit) fractions were analyzed by western blotting using antibodies against the indicated proteins.

 

Figure 4
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  		Fig. 4. Fumarase-MTS point mutations. (A) Growth and complementation of fumarase. MTS point mutants–{Delta}fum1 strain harboring the indicated plasmids encoding MTS point mutants ware diluted and grown on galactose or ethanol-acetate medium plates as indicated. (B) Fumarase-MTS point mutants are processed. MTS point mutants of fumarase were labeled and examined for processing as described in the legend to Fig. 1B. (C) Aconitase-MTS point mutants exhibit alterations in the protein subcellular distribution. Yeast cells expressing fumarase variants were subjected to subcellular fractionation. Equivalent portions from the total (Tot) cytosolic (Cyt) and mitochondrial (Mit) fractions were analyzed by western blotting using antibodies against the indicated proteins.

 

Figure 5
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  		Fig. 5. Aconitase and fumarase and their cytosolic derivates are stable. Yeast strains harboring plasmids encoding the indicated proteins were induced in galactose medium and pulse-labeled with [35S]methionine-cysteine for 15 minutes, followed by a chase (addition of unlabeled methionine-cysteine and cycloheximide) for the times indicated. Total cell extracts were prepared, immunoprecipitated with aconitase or fumarase antiserum and analyzed by SDS-PAGE and autoradiography.

 

Figure 6
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  		Fig. 6. (A) Translocation machinery mutant strains exhibit alterations in protein subcellular distribution. Indicated mutant strains were grown at the permissive temperature. The strains were subjected to subcellular fractionation. Equivalent portions from the total (Tot) cytosolic (Cyt) and mitochondrial (Mit) fractions were analyzed by western blotting using antibodies against the indicated proteins. (B) Growth at low temperature increases the aconitase cytosolic presence. Wild-type yeast cells were grown at 20°C or at 30°C and subjected to subcellular fractionation as described above.

 

Figure 7
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  		Fig. 7. Translocation of aconitase-MTS mutants is slower. Yeast cells expressing the indicated plasmids were incubated for 1 minute with CCCP followed by labeling for 15 minutes with [35S]methionine (in the presence of CCCP). DTT, an excess unlabeled methionine-cysteine and cycloheximide were added (start of the chase). Aliquots were taken at the indicated times, immunoprecipitated with aconitase antiserum and analyzed by SDS-PAGE. Arrows show positions of precursor (p) and mature proteins (m).

 

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