A topological study of the human gamma-glutamyl carboxylase

gamma-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate, In an attempt to de lineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagg ed full-length carboxylase, expressed in 293 cells, it was demonstrated tha t the amino-terminus of the cc is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side, In addition, a series of fusi ons were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algor ithm TOPPRED II. Following in vitro translation of each fusion in the prese nce of canine microsomes, the topological orientation of the Lep tag was de termined by proteinase K digestion and endoglycosidase H (Endo H) cleavage, From the topological orientation of the Lep tag in each fusion, the cc spa ns the ER membrane at least 5 times, with its N-terminus in the cytoplasm a nd its C-terminus in the lumen. (C) 2000 by The American Society of Hematol ogy.