Minisymposium 13: Membrane Transport

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Abs # M1302: Electrophysiological characterization of Arabidopsis OPT oligopeptide transporters

Presenter:	Gassmann, Walter       Contact Presenter
Authors	Osawa, Hiroki  (A) (B)  Pike, Sharon M. (A)   Patel, Ami  (A)   Stacey, Gary  (A) (C)  Gassmann, Walter  (A)
Affiliations:	(A): Division of Plant Sciences, University Of Missouri (B): Graduate School of Agricultural and Life Sciences, University of Tokyo (C): Division of Biochemistry, University of Missouri

The OPT (oligopeptide transporter) class of peptide transporters is found in plants, fungi, bacteria and archaea, but not in animals. The Arabidopsis genome contains nine OPT genes which are thought to function in the transport of oligopeptides and derivatives of 3-6 amino acids in length. However, the transport mechanisms and biological functions of OPTs are poorly understood. To address this, we expressed AtOPTs in Xenopus laevis oocytes for two-electrode voltage clamp studies. We included the Saccharomyces cerevisiae oligopeptide transporter ScOPT1 because of its fairly well defined substrate specificity from previous yeast uptake studies. ScOPT1 produced inward currents in response to glutathione, the phytochelatin PC2, and oligopeptides including GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. AtOPT4 expression was initially poor and was optimized by swapping the 5'-untranslated region with that of ScOPT1. AtOPT4 transported several oligopeptides and displayed highest affinity for KLGL, whereas glutathione and PC2 were not substrates. AtOPT4-mediated KLGL transport was also pH-dependent. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled oligopeptide transporters with broad, but distinct substrate specificities. This is the first electrophysiological characterization of members of this class of transporters from any organism. We have subsequently optimized expression in oocytes of additional AtOPTs and found that AtOPT6 transports KLGL, KLLLG and glutathione. Recent findings will be presented. Identification of possible substrates for AtOPTs is essential for developing hypotheses regarding the biological function of this class of transporters.