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

Anke Reinders,Ye Sun,John M Ward,Kayla L Karvonen

doi:10.1074/jbc.m112.372888

Abstract

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Highlights

Type II sucrose transporters (SUTs) are more selective than type I SUTs
Type I SUTs are less selective for sucrose and transport a broad range of ␣- and ␤-glucosides including the coumarin ␤-glucoside esculin [11]
The type I SUT StSUT1 from potato [28] and the type II SUT OsSUT1 from rice [9] were expressed in yeast, and the cells were incubated in pH 4.0 buffer containing 1 mM esculin, washed, and observed under a fluorescence microscope

Summary

Background

Type II sucrose transporters (SUTs) are more selective than type I SUTs. Results: OsSUT1 was modified using gene shuffling. Type I SUTs are localized to the plasma membrane and are only found in eudicots They have a broad substrate specificity that includes sucrose, maltose, and a variety of ␣- and ␤-glucosides [10, 11]. We hypothesized that conserved residues in type II SUTs that are different in type I SUTs produce the difference in substrate specificity that has been observed To test this experimentally, we used a synthetic version of rice type II sucrose transporter OsSUT1 (Os03g07480) in which 62 amino acid positions were changed to their differentially conserved counterpart present in. OsSUT1 Substrate Specificity type I SUTs. We used gene shuffling [22, 23] of the synthetic coding sequence and a wild-type OsSUT1 coding sequence to produce a library of recombinants and screened these by expression in yeast for the ability to take up esculin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION