SUBCELLULAR-DISTRIBUTION OF SMALL GTP-BINDING PROTEINS IN THE INTESTINAL-CELL LINE CACO-2

The present study describes the subcellular distribution of low molecu lar weight GTP-binding proteins in the human carcinoma cell line Caco- 2. Highly enriched subcellular fractions of basolateral and brush bord er membranes were prepared by differential density centrifugation and divalent cation precipitation. Small molecular weight GTP-binding prot eins were identified after SDS-PAGE transfer to nitrocellulose using [ alpha(32)P]-labelled GTP. Smg-proteins with molecular masses of 28, 27 , 25 and 24 kDa were detectable in all fractions. Homogenate and brush border membrane fraction showed specific binding of [alpha(32)P] GTP to proteins of a molecular mass of 21 kDa, while in the membrane fract ions (apical, basolateral) a high enrichment of 24 kDa smg-proteins wa s detectable. Western blot analysis identified one of the 21 kDa prote ins of the brush border membrane as rhoA. The homogenate of 4, 8, 11 a nd 14 days old Caco-2 cells showed different [alpha(32)P]-GTP binding to 21, 27 and 28 kDa proteins. In conclusion, this study is the first showing the presence and asymmetrical distribution of smg-proteins amo ng the various membrane components in the human carcinoma cell line Ca co-2.