Anti-calreticulin Antibody Binds to a Membrane Protein in Caveolae

Abstract

Caveolae have been reported to contain a number of proteins related to Ca²⁺ mobilization, including an inositol 1,4,5-trisphosphate receptor-like protein, and a plasmalemmal calcium pump. In search of other Ca²⁺-related proteins, we found that a polyclonal antibody to calreticulin (CRT), an endoplasmic reticulum (ER) Ca²⁺-binding protein, binds to the cytoplasmic surface of caveolae. By immunofluorescence microscopy of cultured fibroblasts, labeling by the anti-CRT antibody was found colocalized with caveolin-1. Immunogold electron microscopy of ultrathin frozen sections showed that the antibody decorates the cytoplasmic surface of caveolae. The caveolar labeling could be due to CRT that leaked out of the ER, but the labeling persisted even when isolated plasmalemmal preparations were treated with 10 mM EDTA, 1 M NaCl, or 0.1 M Na₂CO₃ (pH 11). Furthermore, in SDS-digested freeze-fracture replicas, labeling was associated with caveolae in the P face of the plasma membrane. However, in cells transfected with hemagglutinin (HA)-tagged cDNA, the labeling by anti-HA was observed only in the ER and was not found in caveolae. Taken together, the results suggest that a protein structurally similar to CRT is localized in caveolae as an integral membrane protein, and that the protein could be functionally related to the intracellular Ca²⁺ homeostasis.