Phylogenetic analysis of genomic sequences
Genomic DNAs of the following species were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, Wiltshire, UK): Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates sp., Papio hamadryas, Macaca mulatta, Chlorocebus aethiops, Aotus trivirgatus and Saguinus oedipus . Human genomic DNA as a positive control was extracted from HeLa cells according to . Genomic DNA from C57 BL6/J mice was kindly provided by Dr Manuela Wuelling (University of Duisburg-Essen, Essen, Germany). All genomic DNAs were amplified with a REPLI-g whole genome amplification kit (Qiagen, Hilden, Germany) prior to PCR. PCRs were performed using the primers 5'-cggctttcaggcatttgtttag-3' and 5'-tttccccgcttttccagaaccact-3' for amplification of exon 1 and 5'-gtcagacacattctatgtgaaaaac-3' and 5'-cccttgcctggctttatcttcact-3' for exon 3, respectively. PCR products were QIAquick gel extracted (Qiagen, Hilden, Germany), TOPO-TA cloned (Invitrogen, Karlsruhe, Germany) and sequenced (GATC Biotech, Konstanz, Germany). Sequences were edited and analyzed using the BioEdit software . The Ensembl genome browser entries from human [Ensembl:ENSG00000102309], macaque [Ensembl:ENSMMUG00000012956], mouse [Ensembl:OTTMUSG00000018308] and chimpanzee [Ensembl:ENSPTRG00000022022] (exon 3 only) were used as reference sequences .
Parvulin exon 1 and exon 3 sequences obtained in this study have been deposited at EMBL with the following accession numbers: Homo sapiens, [EMBL:AM420633]; Pan troglodytes, [EMBL:AM420634]; Gorilla gorilla, [EMBL:AM420635]; Pongo pygmaeus, [EMBL:AM420636]; Hylobates sp., [EMBL:AM420637]; Papio hamadryas, [EMBL:AM420638]; Macaca mulatta, [EMBL:AM420639]; Chlorocebus aethiops, [EMBL:AM420640]; Aotus trivirgatus, [EMBL:AM420641]; Saguinus oedipus, [EMBL:AM420642]. Chlorocebus aethiops is denoted as Cercopithecus sp. within the respective database entry.
Cell fractionation and Western blotting
For cell fractionation, HeLa cells were grown to confluence in petri dishes in medium containing D-MEM, 1× MEM and 1% FCS (all Gibco/Invitrogen, Karlsruhe, Germany) at 37°C and 7.5% CO2, trypsinized and washed in D-MEM. Cell pellets were separated into cytoplasmic, nucleic, membrane and mitochondrial fractions using the Mitochondria Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The nucleic fraction was resuspended in 200 mM Tris-HCl buffer, pH 8.8. Equal amounts of protein (30 μg) were separated on 15% SDS-PAGE gels and blotted on nitrocellulose membranes (Invitrogen, Karlsruhe, Germany) at 100 mA, 35 min. Following 30 min blocking with TBS-T 150 + 2% (w/v) dry milk, membranes were incubated overnight at 4°C with one of the following primary antibodies in TBS-T 150 + 2% milk: rabbit antiserum against the N-terminal Par17 extension (Ab-EXT, , 1:200 dilution); mouse anti-cytochrome c (clone 7H8.2C12, Abcam, 1:200); rabbit HRP-conjugated anti-GAPDH (Abcam, 1:500). The blots were then incubated with the respective HRP-conjugated secondary antibodies and developed using ECL kits (GE Amersham Bioscience, Freiburg, Germany) and CL-XPosure X-ray film (Perbio Science, Bonn, Germany).
Construction and expression of EGFP fusion proteins
Coding sequences of Par14, Par17-QR, Par17-RS, QR presequence, RS presequence, QR presequence + basic domain and RS presequence + basic domain were PCR amplified using the following primers: 5'-aaaaaagaattcgccaccatgccgcccaaaggaaaaagtggt-3' (Par14 forward); 5'-aaaaaaggatcctttcttccttcgaccataataatatg-3' (Par14 and Par17 reverse); 5'-aaaaaagaattcgccaccatgcccatggcggggcttctaaag-3' (Par17 full length and presequence forward); 5'-atatatggatccttggaagcttgttgttgaacgctg-3' (Par17 presequence reverse); 5'-atatatggattcttgggaccttgagccttcttgtca-3' (Par17 presequence plus basic domain reverse). PCR products were EcoRI/BamHI cloned into the pEGFP-N1 vector (Clontech, Saint-Germain-en-Laye, France) and were verified by sequencing.
For expression of parvulin EGFP constructs HeLa cells were grown on cover slips in 12-well plates in HeLa cell medium (as described above) to about 70–80% confluence. Transfections were performed using FuGene (Roche, Mannheim, Germany) according to the manufacturer's instructions and cells were allowed to grow for 40 h. Then the medium was removed and cells were washed in PBS pH 7.4 (Gibco/Invitrogen, Karlsruhe, Germany). For mitochondrial staining cells were incubated in a 100 nM solution of MitoTracker orange (Molecular Probes/Invitrogen, Karlsruhe, Germany) for 30 min, afterwards washed in PBS and fixed in PBS containing 3% paraformaldehyde and 0.2% Triton X-100 for 10 min. Cover slips were washed and mounted on microscope slides.
Image acquisition and data analysis
Image data were retrieved and analyzed with an Olympus BX61 Microscope equipped with bandpass filter cubes, an UPlan SApo 100× Oil objective, an F-View II camera and the Cell^P software (all from Olympus, Hamburg, Germany, and SIS Soft Imaging Systems, Muenster, Germany). Deconvolution of Z-stacks was performed using the nearest neighbor deconvolution algorithm with a haze removal factor of 99%. Processing of the pictures and calculation of co-localization values were all performed within the Cell^P software. In addition, cells were analyzed with a Leica DM IRB Confocal Microscope with TCS SP 2 Confocal Scanner (Leica, Wetzlar, Germany) to judge the quality of the deconvoluted Z-stacks described above.
Isolation of mitochondria
Mitochondria from yeast were isolated from wild type strain PK82 (MAT α, his4-713, lys2, ura3-52, trp1, leu2-3;  grown in YPG medium (1% (w/v) yeast extract, 2% (w/v) bacto-peptone, pH 5.0, containing 3% (v/v) glycerol) following standard procedures [28, 30]. Rat liver and Jurkat cells mitochondria were obtained essentially as published previously [62, 63].
Import of proteins into isolated mitochondria
Radiolabeled Par17-QR, -RS, Par14 and control proteins were synthesized in rabbit reticulocyte lysate (TNT T7 Coupled Reticulocyte Lysate System, Promega, Mannheim, Germany) in the presence of 35S-labeled methionine (ICN Biomedical Research Products, Eschwege, Germany). In sample sizes of 50–100 μl the radiolabeled proteins were imported into isolated mitochondria as described previously . Under standard conditions, the import assay contained BSA buffer (3% (w/v) BSA, 80 mM KCl, 10 mM MOPS-KOH, pH7.2), 2–5 μl reticulocyte lysate, 2 mM NADH, 1 mM ATP, 20 mM potassium phosphate and yeast mitochondria (30 μg mitochondrial protein). As import into isolated mammalian mitochondria is generally less efficient compared to yeast mitochondria [28, 64], mammalian mitochondria were added at a higher concentration (40 μg mammalian mitochondrial protein). The import reactions were carried out at 25°C and protein uptake stopped by incubation at 0°C. Non-imported proteins were either degraded by addition of proteinase K at a final concentration of at least 100 μg/ml and incubation for 10 min at 0°C or the samples were left untreated. The protease was inactivated by addition of 2 mM PMSF and additional incubation for 5 min at 0°C. Eventually, the mitochondrial membrane potential was dissipated by addition of valinomycin (Sigma, Munich, Germany) at a final concentration of 1 μM from a 100-fold concentrated stock solution in ethanol. Selective opening of the mitochondrial outer membrane was achieved by incubation of mitochondria in EM buffer (1 mM EDTA, 10 mM MOPS-KOH, pH 7.2) for 20 min at 0°C (swelling). Alternatively, digitonin (Calbiochem/Merck, Darmstadt, Germany) was added up to a final concentration of 0.1% (w/v). For association studies, mitochondria were separated from soluble protein by the addition of SEM buffer (500 mM sucrose, 1 mM EDTA, 10 mM MOPS, pH 7.2) and subsequent centrifugation. An additional washing step in a 250 mM sucrose containing SEM buffer was performed to ensure efficient separation of mitochondria from lysate ingredients.
DNA binding assay
HeLa cells were transfected with Par14-, Par17-RS- and Par17-QR-EGFP fusion constructs as described above and lysed in 50 mM K-PO4, 1 mM EDTA, 0.1% Triton X, 1 mM Pefablock (Roth, Karlsruhe, Germany), pH 7.0. Cells were filled up to a total volume of 4 ml with dilution buffer (20 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1% Triton X, 37.5 mM (NH4)2SO4), disrupted by ultrasonic and centrifuged to remove debris (20 min, 4°C, 4000 × g). DNA cellulose (0.25 g calf thymus DNA cellulose (Sigma, Munich, Germany)) was suspended in 3 ml binding buffer (20 mM Tris, pH 7.5, 10% glycerol, 100 mM KCl, 1 mM EDTA, 10 mM NaF and 0.1% Triton X) and incubated rotating at 4°C overnight. A total of 850 μl of cellulose suspension was transferred to a 2 ml microcentrifuge tube, washed with fresh binding buffer and centrifuged (10 min, 4°C, 2000 × g) three times. The supernatant was then replaced by 850 μl of cell lysate. The suspension was incubated with rotating for 1 h at 4°C and then centrifuged (2 min, 10000 × g, 4°C). The supernatant was stored at 4°C. The cellulose was washed successively with binding buffer containing increasing concentrations of KCl. After each washing step the suspension was incubated and centrifuged as described above. All supernatants were methanol/chloroform precipitated, run on 12.5% SDS-PAGE gels and analyzed by Western blotting using anti-GFP antibodies as described above. Blots were analyzed quantitatively by densitometry using Scion Image software .
The experiments were repeated using radiolabeled proteins instead of EGFP fusion constructs. Here, 50 μl of cellulose suspension and 5 μl of cell lysate as well as 50 μl of the different binding buffers were sufficient. All incubation times were reduced to 20 min. Gels were analyzed autoradiographically as described above.