TY - JOUR
T1 - In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins
AU - Nye, Steven H.
AU - Scarpulla, Richard C.
PY - 1990
Y1 - 1990
N2 - To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CATA, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.
AB - To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CATA, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.
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U2 - 10.1128/MCB.10.11.5753
DO - 10.1128/MCB.10.11.5753
M3 - Article
C2 - 2172784
AN - SCOPUS:0025175706
VL - 10
SP - 5753
EP - 5762
JO - Molecular and Cellular Biology
JF - Molecular and Cellular Biology
SN - 0270-7306
IS - 11
ER -