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Title: SITE-DIRECTED MUTAGENESIS OF HIS71 IN THE PROTON-SUCROSE SYMPORTER

Author
item LU, MEI-YEH - PLANT BIOLOGY UOFI URBANA
item SAUER, NORBERT - LEHRTUHL BOTANIK, GERMANY
item BUSH, DANIEL

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: The proton-sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a DEPC-sensitive histidine residue is at or near the substrate-binding site of the symporter (ABB 307:355-360). Among the proton-sucrose symporters identified so far, only His71 is conserved across species and, therefore, is a logical target for detailed investigation. In the results reported here, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Among six different substitutions of His71, H71R transports sucrose at a higher rate than the wild-type symporter. Other substitutions exhibited a range of activities. H71C, for example, resulted in the complete loss of transport capacity while H71Q was almost as active as wild-type. Significantly, H71Q was no longer sensitive to DEPC, suggesting this is the inhibitor-sensitive histidine residue. These data show that His71 is a critical residue for symporter function. We have also identified random mutants in the symporter that alter transport activity. Our aim is to identify essential residues and protein domains as the foundation of a functionally-anchored model of symporter structure with the long-term objective of unraveling the molecular mechanism of the transport reaction.