The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed.
-
Cover Image
Cover Image
Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. ANP, which maintains cardiovascular homeostasis via its diuretic, natriuretic and hypotensive effects, acts through its receptor, GC-A. In this issue of the Biochemical Journal, Miura et al. (pages 1897–1918) established mouse immortalized endothelial cells stably expressing GC-A (SVEC/GC-A) and found that ANP promotes cell spreading and endothelial barrier function via regulation of CCM2, an adaptor protein involved in the pathogenesis of cerebral cavernous malformations, a neurovascular disease characterized by dysfunction of the endothelial barrier. The image shows ANP promotes co-localization of GC-A (green) and CCM2 (red) at membrane ruffles in SVEC/GC-A. Image kindly provided by Koichi Miura
Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p
Mohammad Zulkifli, Anand Kumar Bachhawat; Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p. Biochem J 1 June 2017; 474 (11): 1807–1821. doi: https://doi.org/10.1042/BCJ20161063
Download citation file:
Sign in
Sign in to your personal account
Captcha Validation Error. Please try again.