Functional expression in frog oocytes of human rho 1 receptors produced in Saccharomyces cerevisiae.
The yeast Saccharomyces cerevisiae was engineered to express the rho 1 subunit of the human gamma-aminobutyric acid rho 1 (GABA rho 1) receptor. RNA that was isolated from several transformed yeast strains produced fully functional GABA receptors in Xenopus oocytes. The GABA currents elicited in the oocytes were fast, nondesensitizing chloride currents; and the order of agonist potency was GABA > beta-alanine > glycine. Moreover, the receptors were resistant to bicuculline, strongly antagonized by (1,2,5,6 tetrahydropyridine-4-yl)methylphosphinic acid, and modulated by zinc and lanthanum. Thus, the GABA receptors expressed by the yeast mRNA retained all of the principal characteristics of receptors expressed by cRNA or native retina mRNAs. Western blot assays showed immunoreactivity in yeast plasma membrane preparations, and a rho 1-GFP fusion gene showed mostly intracellular distribution with a faint fluorescence toward the plasma membrane. In situ immunodetection of rho 1 in yeast demonstrated that some receptors reach the plasma membrane. Furthermore, microtransplantation of yeast plasma membranes to frog oocytes resulted in the incorporation of a small number of functional yeast rho 1 receptors into the oocyte plasma membrane. These results show that yeast may be useful to produce complete functional ionotropic receptors suitable for structural analysis.
Martínez-Martínez
Alejandro
A
Laboratory of Cellular and Molecular Neurobiology, Department of Neurobiology and Behavior, University of California, McGaugh Hall 1115, Irvine, CA 92697-4550, USA.
Reyes-Ruiz
Jorge Mauricio
JM
Martínez-Torres
Ataúlfo
A
Miledi
Ricardo
R
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D018079:Receptors, GABA / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014981:Xenopus
D018079:Receptors, GABA; D011993:Recombinant Fusion Proteins