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Mutations in the N ‐terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1 + allows functional expression in Saccharomyces cerevisiae
Author(s) -
Thakur Anil,
Bachhawat Anand K.
Publication year - 2013
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.2939
Subject(s) - mutant , biology , saccharomyces cerevisiae , schizosaccharomyces pombe , mutagenesis , genetics , heterologous expression , gene , wild type , mutation , point mutation , heterologous , biochemistry , recombinant dna
Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe , orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S . cerevisiae . In the present study we employed a genetic strategy to investigate the reason behind the non‐functionality of pgt1 + in S . cerevisiae . Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N ‐terminal region (301–350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1 + proteins not only expressed and localized correctly, but displayed high‐affinity glutathione transport capabilities in S . cerevisae . Comparison of wild‐type pgt1 + with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild‐type pgt1 + in S . cerevisiae . The mRNA levels in wild‐type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem–loop structure appeared to be responsible for inefficient translation in pgt1 + and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild‐type pgt1 + , which led to their functionality in S . cerevisiae . This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system. Copyright © 2012 John Wiley & Sons, Ltd.