The identification of calmodulin-binding sites on mitochondria in cultured 3T3 cells

Cell. 1981 Feb;23(2):533-42. doi: 10.1016/0092-8674(81)90149-5.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Calcium-Binding Proteins / metabolism*
  • Calmodulin / metabolism*
  • Calmodulin-Binding Proteins
  • Carrier Proteins / metabolism*
  • Cell Line
  • Cytoskeleton / metabolism
  • Mice
  • Microtubules / metabolism
  • Mitochondria / metabolism*
  • Mitochondria, Liver / metabolism
  • Molecular Weight
  • Parvalbumins / metabolism
  • Rats
  • Troponin / metabolism
  • Troponin C


  • Calcium-Binding Proteins
  • Calmodulin
  • Calmodulin-Binding Proteins
  • Carrier Proteins
  • Parvalbumins
  • Troponin
  • Troponin C