Amino acid sequence requirements for the association of apocytochrome c with mitochondria.
To examine the amino acid sequence requirements for the biphasic association of Drosophila melanogaster apocytochrome c with mouse liver mitochondria in vitro, recombinant constructs of the protein were prepared. Removal of the C-terminal sequence to residue 58 had little influence, but truncation to residue 50 decreased the association to low levels and removal to residue 36 was even more effective. However, a mutant missing the segment between residues 35 and 66 was fully functional, but, when the C-terminal segment from residue 36 was replaced with a noncytochrome c sequence, the high-affinity phase of the association was lost. A mutant in which residues 90, 91, 92, 96, and 100 were replaced by lysine, leucine, proline, proline, and proline, respectively, to prevent the possible formation of the C-terminal alpha-helix and another mutant in which the C-terminal segment from residue 90 to residue 120 was a noncytochrome c sequence had normal association. In contrast, replacing lysine-5, -7, and -8 by glutamine, glutamic acid, and asparagine, respectively, resulted in loss of the high-affinity phase. The same mutations in the apoprotein lacking the segment between residues 35 and 66 caused, in addition, a decrease of the low-affinity phase association. Thus, the N-terminal region is most critical for apocytochrome c association, but alternative segments of the central and/or C-terminal region can be utilized, where noncytochrome c sequences are ineffective. These results emphasize the wide disparity between the structural requirements for association with mitochondria and for the production of a functional holoprotein.
Sprinkle
J R
JR
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208.
Hakvoort
T B
TB
Koshy
T I
TI
Miller
D D
DD
Margoliash
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000235:genetics* / Q000378:metabolism; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D045304:Cytochromes c; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D051381:Rats
D001059:Apoproteins; D003574:Cytochrome c Group; D045304:Cytochromes c