Strains and plasmids
Experiments were performed in BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), obtained from Euroscarf. Yeast strains BY4741, Δaif1 (YNR074C), Δcpr1, Δcpr2, and Δyca1 were obtained from Euroscarf and were grown on SC medium containing 0.17% yeast nitrogen base (Difco), 0.5% (NH4)2SO4, 30 mg/l of all amino acids (except 80 mg/l histidine and 200 mg/l leucine), 30 mg/l adenine, and 320 mg/l uracil with 2% glucose as carbon source (SCD). For adaptation of protein levels of Aif1p in wild type and Δcpr1 cells we used SCD/G media containing 1% glucose, respectively, 1% galactose for the wild type.
Genomic DNA was isolated from BY4741. To construct Aif1pFLAG
was amplified by PCR, cut with EcoR1 and Not1 and ligated into vector pESC-His (Stratagene). The construct codes for a COOH terminally flag
-tagged protein and was expressed under the control of an inducible Gal1 promoter. Chromosomal COOH terminally yEGFP-tagged AIF1
was generated using the protocol of Knop et al. (1999)
. Vector pYM12 was used as template and kan
MX6-yEGFP-tag cassette was amplified by PCR with primers containing homologous regions to AIF1
. The amplified cassette was transformed into BY4741. To generate an Aif1p/yEGFP fusion under the control of the MET25 promoter, AIF1
was amplified by PCR from plasmid pYCG_YNR074c (EUROSCARF) cut with BamH1–EcoR1 and ligated into vector pUG35.
Test for apoptotic markers
Overexpression of Aif1p with and without hydrogen peroxide for survival assays was performed as described for yeast strain FMY21 (Madeo et al., 2002
), survival platings of Δaif1
strains incubated with or without hydrogen peroxide were performed as described previously for a YCA1
disruptant strain (Madeo et al., 2002
). DAPI staining and TUNEL test were performed as described (Madeo et al., 1997
). To determine the frequency of morphological phenotypes, at least 300 cells of five independent experiments were evaluated. For image acquisition we used a fluorescence microscope (model BH-2RFCA; Olympus), a digital camera (model c35AD-4; Olympus) and analySIS software (Soft Imaging System GmbH). In vivo staining of caspase activity by flow cytometric analysis was performed as described previously (Madeo et al., 2002
Cell extracts were prepared and blots were treated as described previously (Madeo et al., 2002
), probed with murine mAbs against GFP (Roche), murine mAbs against nuclear pore complex proteins (clone MAb414; Convance), or rabbit pAbs against Cox2 (a gift from A. Barrientos, Columbia University, New York, NY).
Staining procedures and epifluorescence microscopy analysis
Chimeric Ynr074-GFP fusion protein localization in mitochondria was shown by colocalization with the vector pYX142 containing a DsRed Su1-69 protein under a TPI promoter (Jakobs et al., 2003
). For determination of localization of the fusion proteins in the nuclei, cells were transformed with vector pUR36-NLS (Rodrigues et al., 2001
), nuclei were in vivo labeled with DsRed-NLS protein. A microscope (model Axiovert 200; Carl Zeiss MicroImaging, Inc.) equipped with filter wheels to control excitation and emission wavelength, with a cooled charge-coupled device (CCD) video camera (model AxioCam HRm; Carl Zeiss MicroImaging, Inc.), and software for image archival and management (AXIOVISION 4) was used for examination of cell populations. The excitation source was a 100 W/DC Hg vapor arc lamp (Carl Zeiss MicroImaging, Inc.). In each case the appropriate fluorescence filter was used.
Spheroblasts and cell fractionation
Spheroblasts were isolated from lyticase-treated cells as described previously (Jazwinski, 1990
). For preparation of nuclei, mitochondria, and cytosolic fraction, cells were inoculated to an OD600nm
= 0.1, and grown on SCD for 4 h. Nuclei were prepared with a 50% percoll gradient as described previously (Shermann et al., 1986
). Preparation of mitochondria was modified according to Daum et al. (1982)
Purification of recombinant Aif1p
AIF1 was expressed from pQE-60 expression vector (QIAGEN) and purified following the manual instructions for purification under denaturing conditions. Refolding was achieved by excessive dialysis against 1 mM EDTA, 0.5 mM EGTA, 50 mM glycine, 0.1% NP-40, 150 mM NaCl, 10% glycerol, and 50 mM Hepes, pH 7.9. After concentration using Centriprep YM-30 (Millipore) Aif1p was stored at −80°C.
Cell extracts were prepared as described previously (Madeo et al., 2002
), with a modified lysis buffer (150 NaCl, 50 mM Tris-HCl, pH 7.4, 5 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM PMSF). To the indicated amount (micrograms) of cell extract 1 μg of plasmid DNA (pESC-His; Stratagene) and 2 mM MgCl2
was added. The reaction mix was incubated for 90 min at 37°C and DNA fragments were separated on a 1% agarose gel.
The indicated amount of purified recombinant Aif1p or BSA was added to 1 μg plasmid DNA (pYES2; Invitrogen), incubated at 30°C for 30 min in the presence or absence of 2 mM MgCl2/CaCl2 followed by gel electrophoresis. Heat-inactivated Aif1p was incubated for 10 min at 100°C. 16 μg of purified recombinant Aif1p or BSA was added to purified yeast nuclei (200 μg of protein) for the indicated time. After incubation the genomic DNA was isolated by incubation in 1% SDS, 1% Triton X-100, 50 mM NaCl, 50 mM MES, pH 6.4, and 0.66 μg proteinase K for 2 h, phenol/chloroform extraction, ethanol precipitation, and incubation in 1 mg/ml RNase. Samples were analyzed by gel electrophoresis.
In vitro mitochondrial import
Radiolabeled preproteins were obtained by in vitro transcription and translation reactions using TNT T7 coupled reticulocyte lysate (Promega) in the presence of redivue l-[35S]methionine (Amersham Biosciences), creating an Aif1p with a NH2 terminally c-Myc tag. Import mixtures contained 1–5% reticulocyte lysate (vol/vol) in 1% BSA (wt/vol), 600 mM sorbitol, 50 mM KCl, 2 mM potassium phosphate, 10 mM MgCl2, 50 mM Hepes-KOH, pH 7.4, 2 mM NADH, and 2 mM ATP, and were incubated with mitochondria (25–50 mg protein) at 25°C for 1 h. Mitochondria were converted to mitoplasts by 10-fold dilution in ice-cold 20 mM Hepes, pH 7.4. Protease treatment was performed by addition of 50 μg/ml proteinase K to the reaction and incubation for 30 min at 0°C. The protease was inactivated by the addition of 1 mM PMSF. Mitochondria or mitoplast were centrifuged at 14,000 g for 15 min at 4°C washed with ice-cold 600 mM sorbitol, 80 mM KCl, 20 mM Hepes, pH 7.4, resuspended in Laemmli buffer, loaded on a 12% SDS polyacrylamide gel, electroblotted onto an Immobilon PVDF membrane (Millipore), and detected by autoradiography.