Association of calmodulin with lysosomes.

ABSTRACT

We examined the subcellular localization of calmodulin in several cultured cells (primary thyroid follicular cells, thyroid C-cell tumour cells (TT), kidney cells (PtK2-L23) and peritoneal macrophages) by indirect immunofluorescence using affinity-purified antibody to calmodulin. When cells were fixed with 3% formaldehyde for 15 min prior to lysis with 0.5% Triton X-100, spindle fibres in mitotic cells were fluorescent and a diffuse cytoplasmic localization of calmodulin was observed in resting cells. However, when cells were lysed with 0.5% Triton X-100 for 90s prior to fixation for 30 min with 3% formaldehyde, three effects were observed. One there was little diffuse cytoplasmic staining. Two discrete vesicles were stained. Three spindle fibres in mitotic cells were fluorescent. The stained vesicles were phase-dense and ranged from 0.1 to 0.5 micron in primary thyroid follicular cells but were smaller in PtK2 and TT cells. The thyroid follicular cells retained vesicular staining after exposure to thyrotropin and isobutylmethylxanthine, but the number of labelled vesicles decreased by almost 80%. Phase-dense vesicles were identified as lysosomes or other acidic vesicles by vital staining with Acridine Orange. After differential centrifugation of thyroid homogenates, calmodulin was measured by radioimmunoassay (RIA) and found in both cytosolic (89%) and membrane vesicle fractions (11%). The vesicular calmodulin was not eluted by washing with 5 mM-EGTA. The thyroid fractions were subjected to SDS-polyacrylamide gel electrophoresis and the gels incubated with 125I-labelled calmodulin to reveal calmodulin acceptor proteins (CAPs). The vesicle fraction contained quantitatively major CAPs with Mr of 200,000, 140,000, 89,000, 38,000 and 34,000, and minor CAPs of 60,000 and 50,000. Washing the pellet with 5 mM-EGTA did not reduce the content of CAPs. Thus, calmodulin and CAPs are present both in the cytoplasm and in a membrane vesicle fraction. The lysosomal locale of calmodulin and the effect of thyrotropin on vesicle number suggest a role for calcium in the regulation of lysosome function.