Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

Abstract

Background: Type II sucrose transporters (SUTs) are more selective than type I SUTs. Results: OsSUT1 was modified using gene shuffling. Variants were selected for the ability to transport the fluorescent ␤-glucoside esculin. Conclusion: Replacement of five amino acids conferred substrate specificity typical of type I SUTs. Significance: The first loop and top of TMS2 and TMS5 are involved in controlling substrate specificity in SUTs.

Conformational Changes Represent the Rate-Limiting Step in the Transport Cycle of Maize SUCROSE TRANSPORTER1

“…Here, we used the competitive inhibitor sucralose to gain a deeper understanding of the mechanism of Suc accumulation in plant cells. The Suc transporter SUT1 from maize, like other clade II SUTs (e.g., rice [Oryza sativa] SUT1 and sugarcane SUT1; Reinders et al, 2012b), shows a higher specificity for Suc than transporters of clade I. Similar to sugarcane SUT1 (Reinders et al, 2012b), transport currents of maize SUT1 were competitively inhibited by the presence of sucralose ( Figure 1D).…”

“…The Suc transporter SUT1 from maize, like other clade II SUTs (e.g., rice [Oryza sativa] SUT1 and sugarcane SUT1; Reinders et al, 2012b), shows a higher specificity for Suc than transporters of clade I. Similar to sugarcane SUT1 (Reinders et al, 2012b), transport currents of maize SUT1 were competitively inhibited by the presence of sucralose ( Figure 1D). Using an elegant gene shuffling method (synthetic template shuffling), the lab of John Ward recently demonstrated that replacement of five amino acids was sufficient to convert a highly specific type II Suc transporter to a less specific type I-like Suc carrier/substrate specificity (Reinders et al, 2012b).…”

“…Similar to sugarcane SUT1 (Reinders et al, 2012b), transport currents of maize SUT1 were competitively inhibited by the presence of sucralose ( Figure 1D). Using an elegant gene shuffling method (synthetic template shuffling), the lab of John Ward recently demonstrated that replacement of five amino acids was sufficient to convert a highly specific type II Suc transporter to a less specific type I-like Suc carrier/substrate specificity (Reinders et al, 2012b). Key residues determining the specificity of type II Mean I tr at 2100 mV pH 4.0 (µA) 23.3 6 0.7 21.9 6 0.3 20.8 6 0.1 22.0 6 0.1 22.0 6 0.1 21.6 6 0.2 K m at 2100 mV pH 5.6 (mM Suc) 10.9 6 1.4 7.7 6 1.4 9.9 6 0.9 6.9 6 0.9 34.7 6 3.8 13.0 6 1.2 pH dependence…”

“…Monosaccharides, such as Glc, Fru, and Man, the disaccharide lactose as well as the trisaccharide raffinose were not transported ( Figure 1A). Thus, maize SUT1 represents a typical member of the type II Suc transporters (Reinders et al, 2012a(Reinders et al, , 2012b. In contrast with Suc, the artificial sweetener sucralose did not evoke macroscopic SUT1-mediated proton currents ( Figure 1A).…”

“…Type II Suc transporters are highly selective for Suc, maltose, salicin, and a-phenylglucoside (Sivitz et al, 2005;Reinders et al, 2006Reinders et al, , 2012bSun et al, 2010). In contrast with type II transporters, type I Suc carriers transport a broad variety of a-and b-glucosides as well as arbutin, esculin, palatinose, and sucralose (Chandran et al, 2003;Sivitz et al, 2005Sivitz et al, , 2007Reinders et al, 2012b). To test the substrate specificity of maize SUT1, belonging to group II, oocytes of X. laevis were injected with SUT1 cRNA and measured with the two-electrode voltage clamp (TEVC) technique after 4 to 5 d of expression.…”