Structure,
function and interaction of the Pho84 and Gtr1 proteins involved in phosphate
uptake in Saccharomyces cerevisiae.
Jens O. Lagerstedt (1), Kent I. Lundh (2), Ian Reeve (3), John C. Voss (3), Bengt
L. Persson (1)
(1) Department of Chemistry and Biomedical Science, Kalmar University, Box 905,
S-391 29 Kalmar; and Department of Biochemistry and Biophysics, Stockholm
University, S-106 91 Stockholm, Sweden (jens.lagerstedt@hik.se); (2) Department
of Chemistry and Biomedical Science, Kalmar University, Box 905, S-391 29
Kalmar, Sweden; (3) Department of Biological Chemistry, University of
California Davis, Davis, CA 95616 USA
The Pho84 protein of Saccharomyces cerevisiae is responsible for the major cellular phosphate uptake under phosphate limiting conditions. This high-affinity phosphate transporter is under such circumstances transcriptionally up-regulated and sorted to the plasma membrane. As external phosphate is totally exhausted the Pho84 is inactivated by endocytosis and routed to the vacuole. Although Pho84 is solely competent in phosphate transport in a reconstituted system, several other proteins are directly or indirectly involved in high-affinity phosphate transport. The G-binding protein Gtr1 is one of those, and has a similar deletion phenotype as the pho84 mutant. The in vivo translation and trafficking of a green fluorescent protein-tagged Pho84 is unaffected by deletion of GTR1, suggesting that the Gtr1 protein exerts its regulation of the high-affinity transport by some other means. To further analyze this and to characterize the structure/function dependency of both Gtr1 and Pho84 we have applied electro paramagnetic spin resonance (EPR) and surface plasmon resonance (SPR) technologies on heterologously produced and affinity-purified proteins. Our studies have revealed a direct interaction between Pho84 and Gtr1, and, furthermore defined the nucleotide dependence of the Gtr1 protein, and a domain-specific dimeric interaction of Gtr1 monomers. In addition, cysteine-scanning analysis of the Pho84 transporter will be discussed.