title: Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.
abstract: Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.
authors.affiliation: Department of Chemical Engineering, Chonbuk National University, 664-14 Deokjin-dong, 1 Ga Deokjin-Gu, Jeonju, 561-756, South Korea.
authors.forename: Jihee
authors.initials: J
authors.lastname: Yoon
authors.affiliation: School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea.
authors.forename: Simranjeet Singh
authors.initials: SS
authors.lastname: Sekhon
authors.affiliation: School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea. kyh@chungbuk.ac.kr.
authors.forename: Yang-Hoon
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Department of Chemical Engineering, Chonbuk National University, 664-14 Deokjin-dong, 1 Ga Deokjin-Gu, Jeonju, 561-756, South Korea. jihomin@jbnu.ac.kr.
authors.forename: Jiho
authors.initials: J
authors.lastname: Min
mesh_terms: D000626:Aminopeptidases / Q000096:biosynthesis* / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015966:Gene Expression Regulation, Fungal; D008247:Lysosomes / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000626:Aminopeptidases; C056860:lysyl aminopeptidase
keywords: 2DE; Aminopeptidase Y; Anticancerous activity; Antimicrobial activity; Lysosomes; S. cerevisiae