Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris.
In Saccharomyces cerevisiae the activity for the lactate-proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a V (max) of 2.1 nmol x s(-1) x mg of dry weight(-1). Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest V (max) (0.84 nmol x s(-1) x mg of dry weight(-1)) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.
Soares-Silva
Isabel
I
Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Schuller
Dorit
D
Andrade
Raquel P
RP
Baltazar
Fátima
F
Cássio
Fernanda
F
Casal
Margarida
M
D019342:Acetic Acid / Q000378:metabolism; D001692:Biological Transport; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D027501:Monocarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010843:Pichia / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D027981:Symporters / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014170:Transformation, Genetic
C118697:JEN1 protein, S cerevisiae; D027501:Monocarboxylic Acid Transporters; D029701:Saccharomyces cerevisiae Proteins; D027981:Symporters; D019344:Lactic Acid; D005947:Glucose; D019342:Acetic Acid