Stimulus-induced association of Ca(2+)-binding proteins with the plasma membrane detected in situ by photolabeling of intact chromaffin and PC12 cells.

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Abstract
To investigate the involvement of cytosolic proteins in exocytosis, a system with high temporal and spatial resolution has been developed that allows us to detect the interaction of Ca2+- and membrane-binding proteins with the plasma membrane during stimulation of intact chromaffin and PC12 (rat pheochromocytoma) cells. We used 5-iodonaphthalene-1-azide (INA), a hydrophobic label that rapidly partitions into the lipid bilayer of biological membranes. Upon photolysis the label covalently attaches to membrane-embedded domains of proteins. Cells, preincubated with INA in the dark, were stimulated by either 300 muM carbamoylcholine or 60 mM K+ and irradiated (20 s) at various time intervals after stimulation. Subsequently, the cytosolic Ca2+- and membrane-binding proteins were isolated in the presence of EGTA (EGTA extract). Of the almost-equal-to 40 proteins in the EGTA extract, 15 (15-100 kDa) are labeled in both cell types. Upon stimulation, labeling is increased up to 3-fold in some of the proteins compared to cells labeled under basal conditions. In the absence of external Ca2+, no increase is observed. The rate of label incorporation is similar to the rate of exocytosis in several of these proteins. These results indicate that in the event of triggered exocytosis some of the Ca2+-binding proteins interact with the plasma membrane and temporarily embed in the lipid bilayer. Our findings support the hypothesis according to which stimulus-induced alterations in the structure of the Ca2+-binding proteins lead to their transient insertion into the membrane and thereby to membrane fusion.

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