IN-SITU SUBCELLULAR-LOCALIZATION OF EPITOPE-TAGGED HUMAN AND RABBIT CARBOXYLESTERASES

Carboxylesterases are a ubiquitous class of enzymes thought to be invo lved in xenobiotic metabolism and detoxification, Primary amino acid s equence data suggest that these proteins localize to the endoplasmic r eticulum, However, since this family of proteins is highly homologous, the generation of specific reagents to monitor expression and. subcel lular localization has been unsuccessful. To accomplish in situ detect ion of a human alveolar macrophage carboxylesterase and a rabbit liver carboxylesterase, we constructed plasmids that expressed recombinant proteins containing an 11 amino acid influenza hemagglutinin tag near the C-terminus. These proteins retained carboxylesterase activity as d etermined by the conversion of o-nitrophenol acetate to o-nitrophenol, Following transfection of plasmids encoding these proteins into mamma lian cells, cells were analyzed by both fluorescence and electron micr oscopy, The tagged enzymes were localized to the endoplasmic reticulum of both Cos7 monkey kidney cells and Rh30 human rhabdomyosarcoma cell s. No tagged protein was detectable in the culture media. Hence, epito pe tagging allowed the analysis of expression and localization of spec ific carboxylesterases, The methods described in this paper are, there fore, applicable to any protein, including those that are highly homol ogous to other candidate molecules. (C) 1998 Wiley-Liss, Inc.