The membrane of the endoplasmic reticulum (ER) is one of the most fluid of all cellular membranes. Hence, the Sarco/Endoplasmic Reticulum Ca²⁺ATPases (SERCAs) are well adopted to function in cholesterol-poor highly fluid membranes. Lipid rafts are thought to first assemble in the Golgi. Thus, the membranes of the Golgi complex are more enriched in cholesterol and therefore are more rigid. Lipid rafts have been characterized by their relative insolubility at low temperatures in detergents such as Triton X-100. Also it has been found that detergent-resistant membranes are enriched in sphingomyelin and cholesterol.

In our study, the microdomain localization of the Secretory Pathway Ca²⁺ATPase 1 (SPCA1) was investigated by Triton X-100 detergent extraction of HT29 cells. Fractions from extracts were analyzed for total cholesterol content and for SPCA1 and marker proteins by Western blotting. Similarly to cholesterol and the raft-resident protein flotillin-2, SPCA1 was found mainly in detergent-resistantfractions, while SERCA3 was detergent soluble. In addition, the time course of solubilization by Triton X-100 was investigated in living COS-1 cells transfected with glycosyl-phosphatidylinositol-anchored (GPI)-CFP, vesicular stomatitis virus G (VSVG)-YFP, SERCA2b-GFP or SPCA1d-GFP. As already reported¹, GPI-CFP was highly resistant to Triton X-100 solubilization, while VSVG-YFP non-raft protein was gradually solubilized. SERCA2b-GFP ER-resident protein also showed a gradual decrease of GFP fluorescence, up to 70% in 3 min. In contrast, SPCA1d-GFP fluorescence decreased by only 10%.

From these results we conclude that SPCA1 is localized in cholesterol-rich and detergent-resistant domains of colon carcinoma cells.

¹Garner et al., (2008) Biophys. J. 94:1326–1340.