scholarly journals Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2585-2593 ◽  
Author(s):  
JA Bristol ◽  
JV Ratcliffe ◽  
DA Roth ◽  
MA Jacobs ◽  
BC Furie ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Vitamin K ◽  
Golgi Complex ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Immunofluorescent Staining ◽  
Secretory Vesicles ◽  
Ovary Cells ◽  
Coagulation Protein

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.

scholarly journals Reconstitution of Brefeldin A–induced Golgi Tubulation and Fusion with the Endoplasmic Reticulum in Semi-Intact Chinese Hamster Ovary Cells

2000 ◽  
Vol 11 (9) ◽  
pp. 3073-3087 ◽  
Author(s):  
Fumi Kano ◽  
Yasushi Sako ◽  
Mitsuo Tagaya ◽  
Toshio Yanagida ◽  
Masayuki Murata
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Tubule Formation ◽  
Intact Cells ◽  
Ovary Cells ◽  
Kinetics Of

The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein–tagged Golgi complex disassembly in streptolysin O–permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in anN-ethylmaleimide–sensitive factor–dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPγS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.

scholarly journals Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells.

1983 ◽  
Vol 97 (6) ◽  
pp. 1777-1787 ◽  
Author(s):  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Vesicular Stomatitis

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.

Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4063-4070
Author(s):  
A J Dorner ◽  
M G Krane ◽  
R J Kaufman
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary ◽  
Heterologous Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Ovary Cells ◽  
Tissue Plasminogen ◽  
Stable Association

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.

scholarly journals N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity

Blood ◽  
2009 ◽  
Vol 113 (4) ◽  
pp. 929-935 ◽  
Author(s):  
Wenhua Zhou ◽  
Han-Mou Tsai
Keyword(s):  
Von Willebrand Factor ◽  
Golgi Complex ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Glycosylation Sites ◽  
Severe Deficiency ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Severe deficiency of ADAMTS13, a plasma metalloprotease, leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains 10 putative N-glycosylation sites in or near its metalloprotease sequence, spacer region, thrombospondin type 1 repeat no. 4 (TSR no. 4), and CUB domains. Tunicamycin treatment markedly decreased the secretion of ADAMTS13 into the culture medium of transfected cells. Nevertheless, the protease was efficiently secreted from N-acetylglucosaminyltransferase I–deficient Lec1 Chinese hamster ovary cells, indicating that N-glycosylation in the endoplasmic reticulum, but not the conversion of oligomannose to complex N-glycans in the Golgi complex, is important for secretion. However, ADAMTS13 with oligomannose N-glycans cleaved its substrate, von Willebrand factor (VWF) multimers, less effectively, with a higher Km but similar kcat value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased secretion only was observed with the N552Q mutant of ADAMTS13. Enzymatic removal of N-glycans from ADAMTS13 did not affect its VWF cleaving activity. Thus, N-glycosylation is necessary for efficient secretion of ADAMTS13, while conversion of the N-glycans from oligomannose to complex type in the Golgi complex enhances the proteolytic activity of the protease toward VWF multimers. After its secretion, ADAMTS13 does not require N-glycans for its VWF cleaving activity.

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