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N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast

AuthorsDrake, Rocío ; Serrano, Aurelio ; Pérez-Castiñeira, J. R.
KeywordsN-terminal signal sequences
Heterologous expression
Saccharomyces cerevisiae
Green fluorescent protein
Proton-translocating inorganic pyrophosphatase
Chimeric proteins
Issue Date2010
PublisherPortland Press
CitationBiochemical Journal 426: 147- 157 (2010)
AbstractExpression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. Here, we show that the expression level and/or subcellular localization in S. cerevisiae of an heterologous type of multispanning membrane protein, the H+-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable Nterminal signal sequences. Chimeric proteins were constructed by adding the putative Nterminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptides of different organisms (from bacteria to plants) and expressed in a yeast conditional mutant deficient in its cytosolic PPi hydrolysis activity when grown on glucose. Chimeric constructs not only substantially enhanced H+- PPase expression levels in transformed mutant cells but also allowed functional complementation in those cases in which native H+-PPase failed to accomplish it. Activity assays and Western blot analyses further demonstrated the occurrence of most H+-PPase in internal membrane fractions of these cells. The addition of N-terminal signal sequences to the vacuolar H+-PPase AVP1 from the plant Arabidopsis thaliana -a protein efficiently expressed in yeast in its natural form- alters the subcellular distribution of the chimeras suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP-H+-PPase fusion proteins.
Identifiersissn: 0264-6021
Appears in Collections:(IBVF) Artículos
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