Technical Reference #1643

1643.

MDR Quinone Oxidoreductases: The Human and Yeast Zeta-Crystallins Sergio Porte; Eva Crosas; Evgenia Yakovtseva; Josep Biosca; Jaume Farres; Rosario Fernandez; Xavier Pares, Universitat Autonoma de Barcelona, Chemico-Biological interaction, 178(1643), (2009)

Abstract:
The medium-chain dehydrogenase/reductase (MDR) superfamily can be divided into Zn-containing andZn-lacking proteins. Zn-containing MDRs are generally well-known enzymes mostly acting as dehydrogenases.The non-Zn MDR are much less studied and classif

Keywords:
zeta0crystallin; enzyme function; human enzyme; quinone oxidoreductase; RNA binding; yeast enzyme

Materials & Methods:
2.1. Protein expression and purification Cloning of human -crystallin and yeast ZTA1DNA heterologous expression in E. coli and protein purification were performed as described [6]. 2.2. Subcellular localization HeLa cells plated onto glass bottom culture dishes (MatTek) were transfected with pEGFP-C2-CRYZ or pAcGFP1-N1-CRYZ using Lipofectamine 2000 (Invitrogen). To confirm mitochondrial localization HeLa cells were cotransfected with pAcGFP1-N1-CRYZ and the mitochondrial marker pDsRed-Mito (Clontech). At 18 h after transfection the medium was replaced by reduced serum medium Opti-MEM and live cells were visualized by using a confocal laser scanning microscope (Leica TCS SP2 AOBS) equipped with a thermostated stage that was kept at 37 ◦C during the course of the experiment. Cells with a high level of expression of green fluorescent protein (GFP) or red fluorescent protein from Discosoma sp. reef coral (DsRed) were selected and imaged. Excitation illumination from argon ion lasers (476 and 488 nm) was used operating on low (middle) power mode and using a 40×1.25 oil-immersion objective and 1.0–2.5 zoom setting. The relative distribution of the protein was estimated by the optical probe technique using the image analysis software equipped with the microscope. The yeast ZTA1-GFP strain and the parental unmodified strain were obtained from Invitrogen Ltd Yeast GFP Clone Collection. For fluorescent analysis both yeast strains were grown to initial stationary phase in rich YPD medium (1% yeast extract 2% peptone 2% glucose) at 30 ◦C and 250 rpm. One to two hours prior to visualization 0.5 g/ml of 4 6-diamidino-2-phenylindole (DAPI) was added to the culture medium for nuclear staining. A Leica-DMRB microscope and 100× PL Fluotar objectivewere used. GFP and DAPI signals were elicited and detected using a Leica 1.3 and A filters respectively. Images were captured with a Leica DC-200 camera driven by DC Viewer software. 2.3. Deletion of the YBR046C gene The coding region fromthe ZTA1 gene (YBR046C)was substituted by the kanMX4 cassette [12] using the one-step gene replacement method [13]. Yeast colonies with the integrated kanMX4 cassette were selected in rich medium YPD (including 2% agar) supplemented with 200 g/ml G418. Positive colonies were verified by PCR analysis. 2.4. Drop-test assay Serial dilutions of yeast cultures grown to exponential phase (to the same cell density) were performed for the deleted zta1 and the wild-type strains. From each dilution 4- l aliquotswere plated on YPD medium containing different drugs. To induce oxidative stress 50 Mor 100 M menadione or 1mM diamide were added to the solid medium. For each experiment a control plate without any treatment was prepared. To test the hydrogen peroxide sensitivity cells were grown to the same cell density and treated with 10mMhydrogen peroxide in the liquidmedia for 1 h prior to being plated on YPD-agar. 2.5. Western-blot analysis of menadione effect on Zta1p expression Yeast cells were grown in the presence or absence of 100 M menadione in minimal defined medium (0.17% yeast nitrogen base without ammonium sulfate 0.5% ammonium sulfate supplemented with complete amino acid Drop-Mix) to 0.7 OD600 units. Immunoblotting was performed with 10 g protein from each yeast extract. An anti-Zta1p polyclonal antibody obtained by standard procedures from rabbit serumwas used in a 1:5000 dilution. The secondary antibody goat anti-rabbit HRP conjugate was from Bio-Rad. As a positive control 10 ng of purified Zta1p were used.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
HeLa