Difference between revisions of "Part:BBa K1470000"
Line 1: | Line 1: | ||
− | |||
− | |||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K1470000 short</partinfo> | <partinfo>BBa_K1470000 short</partinfo> | ||
+ | |||
+ | <html> | ||
+ | <body> | ||
<h4>Natural Function</h4> | <h4>Natural Function</h4> | ||
Line 11: | Line 12: | ||
<p>CAT-1 is a 66 kDa membrane protein. It is built out of up to 622 amino acids contains 14 transmembrane domains which resolutes in seven extracellulare and eight intracellulare domains. There are two sites for N-glycosylation in the third extracellulare loop. The glycosyled position is rather significant for the entering by the virus. The murine leukeamia virus is only able to enter the cell, when it detects the CAT-1 sugar-bound moieties[3].</p><br> | <p>CAT-1 is a 66 kDa membrane protein. It is built out of up to 622 amino acids contains 14 transmembrane domains which resolutes in seven extracellulare and eight intracellulare domains. There are two sites for N-glycosylation in the third extracellulare loop. The glycosyled position is rather significant for the entering by the virus. The murine leukeamia virus is only able to enter the cell, when it detects the CAT-1 sugar-bound moieties[3].</p><br> | ||
− | https://static.igem.org/mediawiki/2014/7/7c/2014Freiburg_Scheme_mCAT-1.jpg | + | <figure><a href="https://static.igem.org/mediawiki/2014/7/7c/2014Freiburg_Scheme_mCAT-1.jpg" width="320" height="240"></a> |
<p>Scheme of mCAT-1. Members of the CAT family are predicted to have 14 transmembrane domains with intracellular N- and C-termini. Two asparagine residues in the third extracellular loop (indicated as branched lines) have been shown to be glycosylated [7].</p><br> | <p>Scheme of mCAT-1. Members of the CAT family are predicted to have 14 transmembrane domains with intracellular N- and C-termini. Two asparagine residues in the third extracellular loop (indicated as branched lines) have been shown to be glycosylated [7].</p><br> | ||
Line 21: | Line 22: | ||
<p>To provide optimal conditions for viral infection, the best point in time for transduction with the largest number of receptors present on the cell surface was determined. On that account HEK-293T cells had been transfected with CAT-1 fused with a HA-tag. Cells expressing CAT-1 were analyzed after distinct incubation times.</p><br> | <p>To provide optimal conditions for viral infection, the best point in time for transduction with the largest number of receptors present on the cell surface was determined. On that account HEK-293T cells had been transfected with CAT-1 fused with a HA-tag. Cells expressing CAT-1 were analyzed after distinct incubation times.</p><br> | ||
− | <figure | + | <figure><a href="https://static.igem.org/mediawiki/parts/1/1f/Freiburg_ha_tag_mcat.png" width="320" height="240"></a> |
</figure> | </figure> | ||
<p>Expression time of the receptor that was transfected into HEK-293T cells. After transfection with mCAT-1-HA cells were lysed with RIPA buffer at distinct points of time. A western blot was performed using an anti-HA antibody.</p><br> | <p>Expression time of the receptor that was transfected into HEK-293T cells. After transfection with mCAT-1-HA cells were lysed with RIPA buffer at distinct points of time. A western blot was performed using an anti-HA antibody.</p><br> | ||
Line 30: | Line 31: | ||
<p>We wanted to detect the localisation of CAT-1 and fused mCherry to its N-terminus. Using confocal microscopy we varified not only the existance of the protein on the cell surface but showed in a spatial way via several sectional planes how the receptore is expressed by HEK-293T cells.</p> | <p>We wanted to detect the localisation of CAT-1 and fused mCherry to its N-terminus. Using confocal microscopy we varified not only the existance of the protein on the cell surface but showed in a spatial way via several sectional planes how the receptore is expressed by HEK-293T cells.</p> | ||
− | <figure | + | <figure><a href="https://static.igem.org/mediawiki/parts/a/a9/Small_Mcat_mcherry.jpg" width="320" height="240"></a> |
</figure> | </figure> | ||
Line 36: | Line 37: | ||
<p>Please click on the link below to view a spatial resolution of CAT-1 in a HEK-293T cell observing the presence of CAT-1 on the surface or inside the cells, like Golgi apparatus or endoplasmic reticulum</p><br> | <p>Please click on the link below to view a spatial resolution of CAT-1 in a HEK-293T cell observing the presence of CAT-1 on the surface or inside the cells, like Golgi apparatus or endoplasmic reticulum</p><br> | ||
− | + | ||
<video src="https://static.igem.org/mediawiki/2014/6/6b/Freiburg2014_confocal_HEK293T_mCAT1.mp4" type="video/mp4" width="480" height="360" autobuffer autoplay loop> | <video src="https://static.igem.org/mediawiki/2014/6/6b/Freiburg2014_confocal_HEK293T_mCAT1.mp4" type="video/mp4" width="480" height="360" autobuffer autoplay loop> | ||
− | </video> | + | </video> |
==References== | ==References== |
Revision as of 19:17, 24 October 2014
Ecotropic murine leukemia virus (MuLV) receptor / Cationic amino acid transporter 1 (CAT-1)
Natural Function
The cationic amino acid transporter 1 (CAT-1) is part of the CAT family which is a subfamily of the solute carrier family 7 (SLC7). These solute carriers are expressed ubiquitously and build the main entry gate for amino acids such as histidine, arginine or ornithin in mammalian cells. They enable the influx of their substrate independent of Na+. Additionally it was shown that the absence of CAT-1 leads to non-viable mice pubs [1][2].
Structure and virus recognition
CAT-1 is a 66 kDa membrane protein. It is built out of up to 622 amino acids contains 14 transmembrane domains which resolutes in seven extracellulare and eight intracellulare domains. There are two sites for N-glycosylation in the third extracellulare loop. The glycosyled position is rather significant for the entering by the virus. The murine leukeamia virus is only able to enter the cell, when it detects the CAT-1 sugar-bound moieties[3].