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 diethyl pyrocarbonate-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, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased V max ) 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 because of alterations in steadystate levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, 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 implicate this region of the protein in the transport reaction.Assimilate partitioning is a fundamental process that allows plants to function as multicellular organisms. In general, oxidized forms of carbon and nitrogen are reductively assimilated in photosynthetic tissues, and then sugars and amino acids are transported in the phloem cells of the plant's vascular system to the heterotrophic organs. In many plants, sucrose is the principal form of translocated photoassimilate and, therefore, it is a central metabolite in plant growth and development. Moreover, sucrose accumulation in the phloem is a pivotal step in partitioning because it produces a large osmotic potential that generates the positive hydrostatic pressure that drives long-distance transport. A key contributor in this system of resource allocation is the proton-sucrose symporter that transports sucrose into the phloem against a large concentration difference (1, 2).The transport properties and bioenergetics of the protonsucrose symporter were initially described in intact tissue systems, and then more detailed analysis was achieved by using purified plasma membrane vesicles and imposed proton electrochemical potential differences (see ref. 2 for review). The symporter is a secondary active carrier that couples sucrose translocation across the plasma membrane to the protonmotive force generated by the H ϩ -pumping ATPase. T...