N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast.
Expression 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. In the present study, we show that the expression level and/or subcellular localization in S. cerevisiae of a heterologous type of multispanning membrane protein, the proton-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable N-terminal signal sequences. Chimaeric proteins were constructed by adding the putative N-terminal 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. Chimaeric 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 demonstrated further 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 chimaeras, suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP (green fluorescent protein)-H+-PPase fusion proteins.
Drake
Rocío
R
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Centro de Investigaciones Cientificas Isla de la Cartuja, Avda. Americo Vespucio, 49, E-41092 Sevilla, Spain.
Serrano
Aurelio
A
Pérez-Castiñeira
José R
JR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015870:Gene Expression; D043564:Inorganic Pyrophosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042541:Intracellular Space / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D021382:Protein Sorting Signals; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; D043564:Inorganic Pyrophosphatase