Author
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CHANG, HUI-CHU - PLANT BIOLOGY UOFI URBANA |
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BUSH, DANIEL |
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Submitted to: Plant Physiology Supplement
Publication Type: Abstract Only Publication Acceptance Date: 8/6/1997 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Amino acids are the predominant form of nitrogen available to the heterotrophic tissues of plants. These essential organic nutrients are transported across the plasma membrane of plant cells by proton-amino acid symporters. Our lab has cloned an amino acid transporter from Arabidopsis, NAT2/AAP1, that represents the first example of a new class of membrane transporters. We are investigating the structure and function of this porter because it is a member of a large gene family in plants and because its wide expression pattern suggests it plays a central role in resource allocation. In the results reported here, we investigated the topology of NAT2 by engineering a c-myc epitope on either the N- or C-termini of the expressed protein. We then used in vitro translation, partial digestion with proteinase K, and immuno-precipitation to identify a family of oriented-peptide fragments. We modeled the topology of NAT2 based on the lengths of the peptide fragments that allowed us to predict the location of protease accessible cleavage sites. We independently identified the location of the N- and C-termini using immunofluorescence microscopy of NAT2 expressed in COS-1 cells. Based on the combined data, we propose an 11 transmembrane domain model with the N-terminus in the cytosol and C-terminus facing outside of cell. This model of protein topology anchors our complementary investigations of porter structure and function using site-directed and random mutagenesis. |
