R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport

Abstract

The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)—a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport.

Graphical abstract

Site-specific replacement of Arg76 in transmembrane region of Asp: Ala antiporter affected transport activities, suggesting the importance of Arg76 for transport function.

Key words:

• amino acid transporter
• liposome
• transport activity
• GxxxG
• substrate transport

Acknowledgments

We thank the Kikkoman Corporation for the gift of the Tetragenococcus halophilus asp operon. We thank Dr. Futai (Graduate School of Agricultural Science, Tohoku University) and Dr. Yoshimura (Liposome Engineering Laboratory, Inc., Graduate School of Engineering, Mie University) for their advice.