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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 973-978
A topological study of the human  -glutamyl carboxylase
Jianke Tie,
Sheue-Mei Wu,
Dayun Jin,
Christopher V. Nicchitta, and
Darrel W. Stafford
From the Department of Biology, Center for Thrombosis and
Homeostasis, University of North Carolina at Chapel Hill, Chapel Hill,
NC, and the Department of Cell Biology, Duke University Medical Center,
Durham, NC.
 -Glutamyl carboxylase (GC), a polytopic membrane
protein found in the endoplasmic reticulum (ER), catalyzes vitamin
K-dependent posttranslational modification of glutamate to
-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.
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