The identification of calmodulin-binding sites on mitochondria in cultured 3T3 cells.
We have uniformly labeled calmodulin with tetramethyl rhodamine isothiocyanate (CaM-RITC) and used the derivative as a molecular probe in order to identify available, unoccupied calmodulin-binding sites. In mildly fixed (3% formalin) cultured 3T3 cells, the biologically active CaM-RITC bound predominantly to mitochondria. Binding was markedly reduced in the presence of 1 mM EGTA. Stelazine, a phenothiozine which binds to calmodulin, prevented the interaction of CaM-RITC with mitochondrial sites. A 10 fold excess of unlabeled CaM competitively inhibited binding. Fluorescently labeled troponin C and parvalbumin did not bind to mitochondria on any other cellular organelle. Rhodamine (TMRITC) alone did not bind to 3T3 mitochondria. Similar results were obtained using 125I-calmodulin binding to isolated rat liver mitochondria. When solubilized mitochondrial proteins were subjected to calmodulin-Sepharose affinity chromatography and eluted with 1 mM EGTA, there were two major polypeptides 120,000 and 67,000 daltons and at least three minor species (100,000, 60,000 and 40,000 daltons). The interaction required an active Ca2+-CaM complex and is specific for CaM. Double fluorescent staining with CaM-RITC and fluorescein-labeled antibodies to tubulin and DNAase I revealed a mitochondrial distribution pattern similar to that of microtubule arrays but unrelated to actin cabling. There was no evidence that CaM-RITC directly interacted with either microtubules or microfilaments.
D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism; D008970:Molecular Weight; D010320:Parvalbumins / Q000378:metabolism; D051381:Rats; D014336:Troponin / Q000378:metabolism; D019209:Troponin C
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D010320:Parvalbumins; D014336:Troponin; D019209:Troponin C