A topological study of the human gamma-glutamyl carboxylase.

Journal Article (Journal Article)

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 delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

Full Text

Duke Authors

Cited Authors

  • Tie, J; Wu, SM; Jin, D; Nicchitta, CV; Stafford, DW

Published Date

  • August 1, 2000

Published In

Volume / Issue

  • 96 / 3

Start / End Page

  • 973 - 978

PubMed ID

  • 10910912

International Standard Serial Number (ISSN)

  • 0006-4971


  • eng

Conference Location

  • United States