In situ subcellular localization of epitope-tagged human and rabbit carboxylesterases.
Carboxylesterases are a ubiquitous class of enzymes thought to be involved in xenobiotic metabolism and detoxification. Primary amino acid sequence data suggest that these proteins localize to the endoplasmic reticulum. However, since this family of proteins is highly homologous, the generation of specific reagents to monitor expression and subcellular localization has been unsuccessful. To accomplish in situ detection 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 determined by the conversion of o-nitrophenol acetate to o-nitrophenol. Following transfection of plasmids encoding these proteins into mammalian cells, cells were analyzed by both fluorescence and electron microscopy. The tagged enzymes were localized to the endoplasmic reticulum of both Cos7 monkey kidney cells and Rh30 human rhabdomyosarcoma cells. No tagged protein was detectable in the culture media. Hence, epitope tagging allowed the analysis of expression and localization of specific carboxylesterases. The methods described in this paper are, therefore, applicable to any protein, including those that are highly homologous to other candidate molecules.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
P M
PM
Potter
J S
JS
Wolverton
C L
CL
Morton
D O
DO
Whipple
M K
MK
Danks
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism* / Q000648:ultrastructure; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D019044:Image Cytometry; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D000939:Epitopes; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases