Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress.
Heart and liver mitochondria contain a structure that is able to form a large non-selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclosporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP. Photolabelling of a 21-kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl-prolyl cis-trans isomerase (PPIase) activity and was inhibited by CSA (Ki, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21-kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane-space marker, adenylate kinase. Photolabelling of the 18-kDa PPIase was unaffected by Ca2+ or ADP. The 21-kDa PPIase was digested with endoproteinase Asp-N and 11 of the peptides were N-terminally sequenced. The sequences were most similar to those of human cyclophilin-D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.
Tanveer
A
A
Department of Biochemistry and Molecular Biology, University College London, England.
Virji
S
S
Andreeva
L
L
Totty
N F
NF
Hsuan
J J
JJ
Ward
J M
JM
Crompton
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000591:Amino Acid Isomerases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins; D016572:Cyclosporine / Q000737:chemistry; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008930:Mitochondria, Liver / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D010777:Photochemistry / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D000081406:Peptidyl-Prolyl Isomerase F; D000244:Adenosine Diphosphate; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; D002118:Calcium