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Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells

¹ Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
² Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan

View larger version (24K): [in a new window]   Fig. 1. Aggregation of mitochondria observed in the temperature-sensitive mtHsp70 mutant cells. (A) Wild-type (SSC1, panels a-c) and the temperature sensitive mtHsp70 mutant (ssc1-2, panels d-f; ssc1-3, panels g-i) cells were grown in YPD at 23°C to an early log phase. Aliquots from the cells grown at 23°C were shifted to 37°C for the periods indicated at the left of the panels. Cells were fixed and prepared for immunofluorescence microscopy with anti-Tom40 antibodies. Bar, 2 µm. (B) Percentage of cells containing aggregated mitochondria plotted against incubation time. At least 150 cells were examined for each measurement. , wild-type (SSC1); , ssc1-2 mutant (ssc1-2); , ssc1-3 mutant (ssc1-3) cells.

View larger version (54K): [in a new window]   Fig. 2. mmm1 mutant cells exhibit spherical mitochondria, which are distinct from the aggregated mitochondria in ssc1 mutant cells. Wild-type (MMM1, panels a,b) and temperature-sensitive mmm1 mutant (mmm1-1, panels c,d) cells were grown in YPD at 23°C to an early log phase. Aliquots from the cells grown at 23°C were shifted to 37°C (panels b,d) for 60 minutes. The cells were fixed and prepared for immunofluorescence microscopy with anti-Tom40 antibodies. Bar, 2 µm.

View larger version (147K): [in a new window]   Fig. 3. Thin-section electron micrographs of yeast cells with aggregated mitochondria. Wild-type (A) and the temperature-sensitive ssc1 mutant cells (ssc1-2, B,C; ssc1-3, D) were grown at 23°C in YPD medium. Aliquots from the cells grown at 23°C were shifted to 37°C for 60 minutes. Cells were prepared for electron microscopy by the permanganate fixation method as described in Materials and Methods. Arrows in panel A show mitochondria. M in panels B-D shows regions where mitochondria cluster. N, nuclei. Bars, 1 µm.

View larger version (23K): [in a new window]   Fig. 4. Cycloheximide treatment did not alleviate mitochondrial aggregation. (A) Wild-type (SSC1) and the temperature sensitive mtHsp70 mutant (ssc1-2 and ssc1-3) cells were grown at 23°C in YPD medium to an early log phase. The culture was split into two and one half received 0.1 mg/ml of cycloheximide (+ CHX). Aliquots were taken at the indicated times after temperature shift to 37°C and cell extracts were prepared, which were then analyzed by SDS-PAGE and immunoblotting using anti-F1ß antibodies. m and p shows the mature and the precursor forms of F1ß, respectively. (B) Wild-type (SSC1, panels a-c) and the temperature sensitive mtHsp70 mutant (ssc1-2, panels d-f, ssc1-3, panels g-i) cells were grown at 23°C in YPD medium to an early log phase. Cycloheximide was added to each culture to 0.1 mg/ml. Aliquots from the cells grown at 23°C were shifted to 37°C for the periods indicated at the left of the panels. Cells were fixed and prepared for immunofluorescence microscopy with anti-Tom40 antibodies. Bar, 2 µm. (C) Percentage of cells containing aggregated mitochondria was plotted against time of incubation at 37°C. At least 150 cells were examined for each time point. , wild type (SSC1); , ssc1-2 mutant (ssc1-2); , ssc1-3 mutant (ssc1-3).

View larger version (25K): [in a new window]   Fig. 5. Mitochondrial morphology in the temperature-sensitive tim23 mutant or in the [rho0] mutant. (A) Wild-type (YPH500/pAK2, panels a,b) and the temperature-sensitive tim23 mutant (SNY1010-3A/pAK2, panels c,d) cells were grown at 23°C in SCGal medium to express the pCOXIV-S65TGFP fusion protein. In panels b,d, cells were incubated at 37°C for 2 hours before fixation. Mitochondrial morphology was observed by GFP fluorescence. Bar, 2 µm. (B) Wild-type (SEY6210/pAK2, panel a) and its isogenic [rho0] mutant (SEY6210 rho0/pAK2, panel b) cells were grown at 23°C and fixed. Mitochondrial morphology was observed by GFP fluorescence. Bar, 2 µm.

View larger version (39K): [in a new window]   Fig. 6. Depletion of Mdj1p causes aggregation of mitochondria. (A) Wild-type (MDJ1, panels a-f) and the mdj1 mutant (mdj1, panels g-l) cells were grown at 23°C in YPD medium to an early log phase. The culture was split into two and one half received 0.1 mg/ml of cycloheximide (+ CHX). Aliquots from the cells grown at 23°C were shifted to 37°C for the periods indicated at the left of the panels. Cells were fixed and prepared for immunofluorescence microscopy with anti-Tom40 antibodies. Bar, 2 µm. (B) Wild-type and the mdj1 mutant cells were grown at 16°C, 23°C or 30°C in YPD medium to an early log phase. The culture was split into two and was incubated for 2 hours with (+ CHX) or without (- CHX) 0.1 mg/ml of cycloheximide. For 37°C, wild-type and the mdj1 mutant cells were grown at 23°C in YPD medium to an early log phase. The culture was split into two and was incubated for 2 hours at 37°C with (+ CHX) or without (- CHX) 0.1 mg/ml of cycloheximide. Cells were fixed and prepared for immunofluorescence microscopy with anti-Tom40 antibodies. Percentage of cells containing aggregated mitochondria was measured and plotted. At least 150 cells were examined for each measurement. (C) Percentage of cells containing aggregated mitochondria was plotted against time of incubation at 37°C. At least 150 cells were examined for each time point. , wild-type cells (MDJ1); , mdj1 mutant cells (mdj1 - CHX); , mdj1 mutant cells treated with cycloheximide (mdj1 + CHX). (D) Wild-type (MDJ1) and the mdj1 mutant (mdj1) cells were grown at 23°C in YPD medium and shifted to 37°C. Two hours after the temperature upshift (indicated by the vertical arrow), the culture was split into two and one half received 0.1 mg/ml of cycloheximide (+ CHX). Cells were then shifted to 23°C and the percentage of cells containing aggregated mitochondria was plotted against time of incubation. At least 150 cells were examined for each time point. , wild type cells (MDJ1); , mdj1 mutant cells (mdj1 - CHX); , mdj1 mutant cells treated with cycloheximide during the recovery period (mdj1 +CHX).

View larger version (36K): [in a new window]   Fig. 7. The actin cytoskelton is normal in temperature-sensitive mtHsp70 mutant and mdj1 cells. Wild-type (a,b), temperature-sensitive mtHsp70 mutant (ssc1-2, panels c,d, ssc1-3, panels e,f ) and mdj1 mutant (mdj1, panels g,h) cells were grown in YPD at 23°C to an early log phase. Aliquots from the cells grown at 23°C were shifted to 37°C for 60 minutes. The cells were then fixed and the actin cytoskelton was stained with rhodamine-conjugated phalloidin. Bar, 2 µm.

View larger version (29K): [in a new window]   Fig. 8. Latrunculin B treatment partially suppressed the mitochondrial aggregation phenotype in the ssc1-2 and the mdj1 mutants. (A) Wild-type, ssc1-2 and mdj1 mutant cells were grown in YPD at 23°C to an early log phase. Latrunculin B was added to 0.2 mM, and the cells were incubated at 37°C for 60 minutes. The cells were then fixed and prepared for immunofluorescence microscopy with anti-Tom40 antibodies. After latrunculin B treatment, cells were classified into those containing branched and tubular (a), aggregated (b), short tubular (c) or small spherical (d) mitochondria. Bar, 2 µm. (B) Percentages of cells containing branched and tubular (a), aggregated (b), short tubular (c) or small spherical (d) mitochondria. At least 100 cells were analyzed.

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