In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells

1998 ◽  
Vol 00 (1) ◽  
pp. 11.3.1-11.3.22
Author(s):  
Bernard B. Allan ◽  
William E. Balch
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Golgi Transport ◽  
Vitro Analysis ◽  
In Vitro Analysis

scholarly journals BAG-6 is essential for selective elimination of defective proteasomal substrates

2010 ◽  
Vol 190 (4) ◽  
pp. 637-650 ◽  
Author(s):  
Ryosuke Minami ◽  
Atsuko Hayakawa ◽  
Hiroki Kagawa ◽  
Yuko Yanagi ◽  
Hideyoshi Yokosawa ◽  
...  
Keyword(s):  
Mhc Class I ◽  
Mammalian Cells ◽  
Human Major Histocompatibility Complex ◽  
Histocompatibility Complex ◽  
Selective Elimination ◽  
Nascent Chain ◽  
Vitro Analysis ◽  
In Vitro Analysis

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.

scholarly journals Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

1995 ◽  
Vol 129 (4) ◽  
pp. 1049-1059 ◽  
Author(s):  
R Kuriyama ◽  
M Kofron ◽  
R Essner ◽  
T Kato ◽  
S Dragas-Granoic ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Amino Acid Residues ◽  
Central Stalk ◽  
Vitro Analysis ◽  
Kinesin Superfamily ◽  
In Vitro Analysis

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

scholarly journals α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

2010 ◽  
Vol 21 (11) ◽  
pp. 1850-1863 ◽  
Author(s):  
Nandhakumar Thayanidhi ◽  
Jared R. Helm ◽  
Deborah C. Nycz ◽  
Marvin Bentley ◽  
Yingjian Liang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Direct Action ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Protein Receptors

Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

The native structure of the eukaryotic ribosome

1983 ◽  
Vol 41 ◽  
pp. 432-433
Author(s):  
R.A. Milligan ◽  
P.N.T. Unwin
Keyword(s):  
Ribosomal Proteins ◽  
Crystal Form ◽  
Native Structure ◽  
X Ray Diffraction ◽  
Eukaryotic Ribosome ◽  
Vitro Analysis ◽  
In Vitro Analysis ◽  
Resolution Structure ◽  
Stable Unit

A detailed understanding of the mechanism of protein synthesis will ultimately depend on knowledge of the native structure of the ribosome. Towards this end we have investigated the low resolution structure of the eukaryotic ribosome embedded in frozen buffer, making use of a system in which the ribosomes crystallize naturally.The ribosomes in the cells of early chicken embryos form crystalline arrays when the embryos are cooled at 4°C. We have developed methods to isolate the stable unit of these arrays, the ribosome tetramer, and have determined conditions for the growth of two-dimensional crystals in vitro, Analysis of the proteins in the crystals by 2-D gel electrophoresis demonstrates the presence of all ribosomal proteins normally found in polysomes. There are in addition, four proteins which may facilitate crystallization. The crystals are built from two oppositely facing P4 layers and the predominant crystal form, accounting for >80% of the crystals, has the tetragonal space group P4212, X-ray diffraction of crystal pellets demonstrates that crystalline order extends to ~ 60Å.

1163: Radial Dilation of Ureteral Balloons: A Comparative in-Vitro Analysis

2005 ◽  
Vol 173 (4S) ◽  
pp. 315-316
Author(s):  
Kari Hendlin ◽  
Brynn Lund ◽  
Manoj Monga
Keyword(s):  
Vitro Analysis ◽  
In Vitro Analysis

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

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