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.[1]References
- The identification of calmodulin-binding sites on mitochondria in cultured 3T3 cells. Pardue, R.L., Kaetzel, M.A., Hahn, S.H., Brinkley, B.R., Dedman, J.R. Cell (1981) [Pubmed]
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