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Title: HIS-65 IS AN ESSENTIAL AMINO ACID IN THE PROTON-SUCROSE SYMPORTER WHOSE MODIFICATION WITH SITE-SPECIFIC MUTAGENESIS INCREASES TRANSPORT ACTIVITY

Author
item LU, JADE - PLANT BIOLOGY UOFI URBANA
item BUSH, DANIEL

Submitted to: Journal of Experimental Biology Online, Vol 3
Publication Type: Abstract Only
Publication Acceptance Date: 8/14/1998
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. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities, for example, the H65C mutant resulted in the complete loss of transport capacity while H65Q was almost as active as wild-type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not due to alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to DEPC inactivation, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding and increased rates of transport activity implicate this region of the protein in the transport reaction.