Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation.
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.
Bristol
J A
JA
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, MA 02111, USA.
Ratcliffe
J V
JV
Roth
D A
DA
Jacobs
M A
MA
Furie
B C
BC
Furie
B
B
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D016466:CHO Cells / Q000378:metabolism; D002262:Carboxy-Lyases / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003594:Cytoplasmic Granules / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D000939:Epitopes / Q000276:immunology; D018698:Glutamic Acid / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011498:Protein Precursors / Q000276:immunology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011516:Prothrombin / Q000096:biosynthesis* / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014812:Vitamin K / Q000502:physiology*
D018076:DNA, Complementary; D000939:Epitopes; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014812:Vitamin K; D018698:Glutamic Acid; D011516:Prothrombin; D002262:Carboxy-Lyases; C022436:preprothrombin carboxylase