Author
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CHANG, HUI-CHU - PLANT BIOLOGY UOFI URBANA |
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BUSH, DANIEL |
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Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 11/1/1997 Publication Date: N/A Citation: N/A Interpretive Summary: Although plants are photosynthetic organisms, many harvested tissues are non-photosynthetic and, therefore, these organs must import sugars and amino acids to grow. Unfortunately, the amount of certain amino acids present in many organs limits the nutritional value of several of our most important crops, including barley and corn. Generally, inorganic nitrogen is incorporated into amino acids in leaves and then transported to the edible parts of the plant such as fruits, grains, and tubers. Imported amino acids are essential not only to support growth and development of the harvested tissue; they also determine the nutritional value of edible tissues for humans and live stock. One strategy for enhancing nutritional value of some crops is to alter the amount of amino acids transported to harvested tissues. We have discovered and isolated several plant genes that encode high affinity amino acid transporters. The results reported here provides important information about the structure and function of one of these carriers. This is a vital first step in using biotechnology to improve the amino acid content of nutritionally limited agricultural crops. 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. An amino acid transporter has been cloned by our lab 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 epitome on either the NO or C-terminus of the expressed protein. We then used in vitro translation, partial digestion with proteinase K, and immunoprecipitation to identify a group 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 a novel 11 transmembrane domain models with the N-terminus of the protein inside the cytosol and C-terminus facing outside the cell. |
