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Biosynthesis of prothrombin: intracellular localization of the vitamin
K-dependent carboxylase and the sites of gamma-carboxylation
JA Bristol, JV Ratcliffe, DA Roth, MA Jacobs, BC Furie and B Furie
Center for Hemostasis and Thrombosis Research, New England Medical Center,
Boston, MA 02111, USA.
Prothrombin is a vitamin K-dependent blood coagulation protein that
undergoes posttranslational gamma-carboxylation and propeptide cleavage
during biosynthesis. The propeptide contains the gamma-carboxylation
recognition site that directs gamma-carboxylation. To identify the
intracellular sites of carboxylation and propeptide cleavage, we monitored
the synthesis of prothrombin in Chinese hamster ovary cells stably
transfected with the prothrombin cDNA by immunofluorescent staining. The
vitamin K-dependent carboxylase was located in the endoplasmic reticulum
and Golgi complex. Antibodies specific to prothrombin processing
intermediates were used for immunocytolocalization.
Anti-des-gamma-carboxyprothrombin antibodies stained only the endoplasmic
reticulum whereas antiproprothrombin antibodies (specific for the
propeptide) and antiprothrombin:Mg(II) antibodies (which bind the
carboxylated forms of proprothrombin and prothrombin) stained both the
endoplasmic reticulum and the Golgi complex.
Antiprothrombin:Ca(II)-specific antibodies (which bind only to the
carboxylated form of prothrombin lacking the propeptide) stained only the
Golgi complex and secretory vesicles, and colocalized with antimannosidase
II and anti-p200 in the juxtanuclear Golgi complex. These results indicate
that uncarboxylated proprothrombin undergoes complete gamma-carboxylation
in the endoplasmic reticulum and that gamma-carboxylation precedes
propeptide cleavage during prothrombin biosynthesis.
Volume 88,
Issue 7,
pp. 2585-2593,
10/01/1996
Copyright © 1996 by The American Society of Hematology
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