B. R. Brinkley

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.

FLUORESCENTLY‐LABELED CALMODULIN LOCALIZES SPECIFIC SITES ON MITOCHONDRIA

Calmodulin (CaM) has been implicated as a calcium mediator of several cellular processes including motility, secretion, and chromosome movement. It exerts its effect through a series of activation steps, initiated by the binding of calcium. The induced change in molecular conformation allows CaM to interact with enzymes and other binding proteins. These and other biochemical reactions occur in concert to bring about the proper physiological response. Intracellular localization of CaM, CaM-regulated enzymes and CaM binding proteins reveal areas of CaM activity, as in the mitotic apparatus (Welsh et al. 1979. Proc. Natl. Acad. Sci. USA 75: 1867) and the postsynaptic density (Lin et al. 1980. 1. Cell Bio. 85: 473). Studies using immunochemical techniques identify binding sites occupied by endogenous calmodulin. Such observations are an aid in correlating known biochemical reactions with particular cellular activities. We have directly labeled CaM with rhodamine in order to identify available, unoccupied CaM binding sites. The fluorescent conjugate (CaM-RITC) retained full ability to activate CAMP phosphodiesterase. In mildly fixed cultured 3T3 cells (3% formalin) the biologically active CaM-RITC bound predominantly to mitochondria (FIGURE 1A). Binding was markedly reduced in the presence of 1 mM EGTA (FIGURE IB]. Stelazine. a phenothiozine which binds to calmodulin, prevented the interaction of CaM-RITC with mitochondria1 sites. In addition, a ten-fold excess of unlabeled CaM competitively inhibited binding. Fluorescently labeled troponin C and parvalbumin did not bind to mitochondria or any other cellular organelles. Rhodamine (TMRITC) alone did not bind to 3T3 mitochondria. Thus, the interaction observed in FIGURE 1A requires an active Ca2+-CaM complex and is specific for CaM. The physiological role of CaM sites associated with mitochondria is not presently understood. Our studies suggest that CaM may regulate outer mitochondria1 membrane enzymes. Further evidence suggests the possibility of differential occupation of these sites during varying metabolic states of the cell. It is anticipated that microinjection of CaM-RITC into hormonally responsive living cells will further elucidate the cellular role of the CaMmitochondria interaction.