Targeting of cloned firefly luciferase to yeast mitochondria.
The firefly luciferase gene (luc) was fused to a 5' fragment of the 70-kDa protein gene (70K) from yeast. The fragment codes for the N-terminal putative signal sequence which targets and anchors the 70-kDa protein to the cytoplasmic side of the outer membrane in mitochondria. Two versions of the fusion gene, 70K[232]::luc and 70K[93]::luc (containing 292 and 93 5' codons from 70K, respectively), were constructed in a bacterial expression plasmid. Both the genes were expressed in Escherichia coli, and in both cases, bioluminescence activity was associated with the expression. The 70K[93]::luc gene was transferred to a yeast-bacteria shuttle vector used to transform Saccharomyces cerevisiae cells. As a control, the same strain was transformed with a plasmid including the original luc. With both transformants, bioluminescence activity was detected in intact cells and crude extracts. Upon growth on a nonfermentable carbon source and fractionation, the product of the fusion gene was associated mostly with mitochondria. In the control transformant, the product of luc was more delocalized. However, a significant amount remained associated with isolated mitochondria. No such spontaneous association of purified luciferase with wild-type mitochondria was observed in vitro. Trypsin treatment of mitochondria isolated from both transformed strains indicated that the fusion protein is anchored to the outer membrane and exposed to the medium while the unfused luciferase retained with the mitochondria is occluded in a compartment unaccessible to trypsin and released in the presence of detergent. The fusion protein retained the major catalytic properties of the parent firefly luciferase, as determined in solution.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
D000255:Adenosine Triphosphate / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008156:Luciferases / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D008156:Luciferases