In vivo mitochondrial import. A comparison of leader sequence charge and structural relationships with the in vitro model resulting in evidence for co-translational import.
The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Heard
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Weiner
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D000444:Aldehyde Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism
D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000444:Aldehyde Dehydrogenase