title: Yeast cell-based analysis of human lactate dehydrogenase isoforms.
abstract: Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China and.
authors.forename: Lulu Ahmed
authors.initials: LA
authors.lastname: Mohamed
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tachikawa
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China and xdgao@jiangnan.edu.cn.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Gao
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China and hideki@jiangnan.edu.cn.
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakanishi
mesh_terms: D055162:Biocatalysis; D057075:Enzyme Assays; D005285:Fermentation; D005816:Genetic Complementation Test; D057166:High-Throughput Screening Assays; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D000080864:Lactate Dehydrogenase 5; D019344:Lactic Acid / Q000378:metabolism; D008164:Luminescent Proteins; D019289:Pyruvic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; D019344:Lactic Acid; D008164:Luminescent Proteins; D019289:Pyruvic Acid; D000097573:Red Fluorescent Protein; C409667:lactate dehydrogenase 1; C409673:lactate dehydrogenase C4
keywords: S. cerevisiae; alcoholic fermentation; lactate dehydrogenase; lactic acid fermentation; pyruvate decarboxylase