Yeast strains and growth media
Standard genetic techniques were used for growth and manipulation of yeast strains. Unless stated otherwise, the wild-type strains YPH499 and W303 were used. For construction of ugo1Δ
mutant strains in W303 background, the UGO1
genes were deleted by PCR-mediated gene replacement with kan
MX4 and HIS3
-MX6 cassette, respectively. The mas37Δ
(Habib et al., 2005
) and tim8Δ/tim13Δ
(Paschen et al., 2000
) strains were previously described. mim1Δ
+MIM1ΔN, and mim1Δ
+MIM1−FL strains were constructed as reported by Popov-Celeketić et al. (2008)
. The tom70Δ/tom71Δ
double deletion (Kondo-Okamoto et al., 2008
) and TIM10-1
(Koehler et al., 1998
) strains were gifts from K. Okamoto (Osaka University, Osaka, Japan) and C. Koehler (University of California, Los Angeles, Los Angeles, CA), respectively. Transformation of yeast was performed according to the lithium-acetate method. Yeast cells were grown under aerobic conditions in yeast peptone dextrose, YPGal (1% yeast extract, 2% bactopeptone, and 2% galactose), Lac, synthetic dextrose–Trp, or synthetic dextrose–Ura media.
Recombinant DNA techniques
To obtain a C-terminally HA-tagged Ugo1, a sequence encoding 2× HA was PCR amplified from the pFA6a-3HA-KanMX4 vector and inserted into the target vector pYX113 using SalI and XhoI restriction sites. UGO1 without its stop codon was amplified via PCR from genomic DNA isolated from the YPH499 strain and introduced into the modified vector using EcoRI and SalI restriction sites. For cell-free experiments, this construct (pYX113 UGO1-2HA) was used as a template for PCR amplification of UGO1-2HA. PCR product obtained in this way was inserted into pGEM4 vector by use of the SmaI and XbaI restriction sites. The cytosolic domain of Tom70 (Δ amino acid residues 1–34) was amplified by PCR and introduced into the pGEX4T vector using the BamHI and HindIII restriction sites.
Mitochondria were isolated from yeast cells by differential centrifugation as previously described (Daum et al., 1982
). For swelling experiments, isolated mitochondria were incubated with a hypotonic buffer (20 mM Hepes, pH 7.2) for 30 min on ice. Then, it was supplemented with urea to a final concentration of 1 M and incubated on ice for a further 5 min. Swollen mitochondria were reisolated by centrifugation and resuspended in import buffer. Chemical amounts of Ugo1-HA were produced in wheat germ lysate according to the manufacturer´s instructions (RTS 100 Wheat Germ CECF kit; 5Prime). The recombinant proteins GST, GST-Tom70 (cytosolic domain), MBP, and MBP-Mim1 were expressed in Escherichia coli
BL21 cells as soluble proteins. Purification of recombinant proteins was performed by affinity chromatography according to the manufacturer´s instructions using either glutathione beads (Macherey-Nagel) or maltose-coupled beads (New England Biolabs, Inc.).
Protein samples were analyzed by SDS-PAGE and blotting to nitrocellulose membranes followed by visualization through autoradiography. Alternatively, incubation with antibodies was performed according to standard procedures, and visualization was performed via the ECL method. The antibody against Ugo1 was a gift from S. Hoppins and J. Nunnari (University of California, Davis, Davis, CA). Intensity of the observed bands was quantified with automatic imaging data analysis software (raytest GmbH). Unless stated otherwise, each presented experiment represents at least three repetitions.
In vitro protein import
Import experiments with radiolabeled precursor proteins and isolated mitochondria were performed in an import buffer containing 250 mM sucrose, 0.25 mg/ml BSA, 80 mM KCl, 5 mM MgCl2, 10 mM MOPS-KOH, 2 mM NADH, and 2 mM ATP, pH 7.2. Radiolabeled precursor proteins were synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine. In some cases, the import reaction was treated with 0.3 U/µl apyrase. Trypsin treatment of mitochondria was performed by adding 50 µg/ml trypsin for 25 min on ice. Trypsin was then inhibited by adding 1.5 mg/ml soybean trypsin inhibitor for 10 min on ice. For blocking the TOM complex import pore, the recombinant precursor protein pSu9-DHFR was added to 30 µg of isolated mitochondria immediately before the import reaction. In the carbonate extraction reaction, mitochondria were dissolved in 0.1 M Na2CO3. After 30 min on ice, the sample was centrifuged (75,000 g for 30 min at 2°C), and pellet and supernatant were analyzed.
Mitochondria were lysed in 40 µl digitonin-containing buffer (1–1.5% digitonin, 20 mM Tris-HCl, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, and 1 mM PMSF, pH 7.4). After incubation for 15 min at 4°C and a clarifying spin (30,000 g
for 15 min at 2°C), 5 µl of sample buffer (5% [weight/volume] Coomassie brilliant blue G-250, 100 mM Bis-Tris, and 500 mM 6-aminocaproic acid, pH 7.0) was added, and the mixture was analyzed by electrophoresis in a 6–13% gradient BN gel (Schägger et al., 1994
). Gels were blotted to polyvinylidene fluoride membranes, and proteins were further analyzed by autoradiography or immunodecoration.
Online supplemental material
Fig. S1 presents data supporting the proposal that the N-terminal domain of Mim1 is not required for optimal biogenesis of Ugo1. Fig. S2 shows that the biogenesis of Ugo1 does not require the TOM import pore or elements in the IMS. Fig. S3 includes results of experiments demonstrating that the insertion of Ugo1 is independent of the TOB complex. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201102041/DC1