Role of Tim17 Transmembrane Regions in Regulating the Architecture of Presequence Translocase and Mitochondrial DNA Stability

Abstract

Mitochondrial life cycle and protein import are intricate cellular processes, which require precise coordination between the transport machineries of outer and inner mitochondrial membranes. Presequence translocase performs the indispensable function of translocating preproteins having N-terminal targeting sequences across the inner membrane. Tim23 forms the core of the voltage-gated import channel, while Tim17 is presumed to maintain the stoichiometry of the translocase. However, mechanistic insights into how Tim17 coordinates these regulatory events within the complex remained elusive. We demonstrate that Tim17 harbors conserved G/AXXXG/A motifs within its transmembrane regions and plays an imperative role in the translocase assembly through interaction with Tim23. Tandem motifs are highly essential, as most of the amino acid substitutions lead to nonviability due to the complete destabilization of the TIM23 channel. Importantly, Tim17 transmembrane regions regulate the dynamic assembly of translocase to form either the TIM23 (PAM)-complex or TIM23 (SORT)-complex by recruiting the presequence translocase-associated motor (PAM) machinery or Tim21, respectively. To a greater significance, tim17 mutants displayed mitochondrial DNA (mtDNA) instability, membrane potential loss, and defective import, resulting in organellar dysfunction. We conclude that the integrity of Tim17 transmembrane regions is critical for mitochondrial function and protein turnover.

Keywords: membrane potential; mitochondria; mtDNA stability; preprotein import; presequence translocase.

FIG 1

Isolation of Ts mutants from different transmembrane regions of Tim17 protein. (A) Growth phenotype analysis. Wild-type (WT) and tim17 mutant strains isolated from transmembrane regions (TM1, TM2, TM3, and TM4) were allowed to grow until mid-log phase in liquid YPD medium at 30°C. Yeast cells corresponding to an A₆₀₀ of optical density at 600 nm (OD₆₀₀) of 0.5 were harvested, serially diluted, and spotted onto YPD or YPG medium. Plates were incubated at the indicated temperatures, and images were captured after 72 h. (B) Growth phenotypes of double tim17 mutants. Mutants from tandem G/AXXXG/A motifs were spotted onto medium supplemented with 5-fluoroorotic acid (5-FOA) and incubated at 30°C, and images were captured after 72 h. (C) Modular representation of Tim17 protein. The topology of different helices and loops and the positions of the amino acids spanning each of the segments of the Tim17 protein are highlighted. Amino acid positions of the Ts mutants from TM1, TM2, and TM3 and the lethal mutant from the TM4 region are indicated.

FIG 2

Measurement of steady-state protein levels and phenotypic analyses of transmembrane regions (TM1 to -3) mutants of Tim17. (A to C) Protein expression analysis. Steady-state protein levels of the wild type and tim17 mutants (from TM1, TM2, TM3) were analyzed by Western blotting using specific antibodies for different components of presequence translocase. (D) Analysis of protein half-lives. Wild-type and mutant cells were treated with cycloheximide (50 μg/ml) to inhibit the protein translation, and cells were collected at different time intervals. Lysates were prepared and analyzed by Western blotting (AU, arbitrary units). The amounts of different proteins were quantified by densitometry using ImageJ software. (E) The wild type and tim17 mutants (from TM1 and TM2) expressed under the control of the centromeric TEF promoter were serially diluted and spotted on the indicated media and incubated at different temperatures. (F) Protein overexpression is analyzed for tim17 mutants (TM1 and TM2) using desired specific antibodies by Western blotting.

FIG 3

Analysis of steady-state proteins levels of lethal mutants. (A) C-terminal FLAG-tagged tim17 mutants were transformed into wild-type isolates harboring endogenous Tim17 (pRS-316 Tim17), and expression levels of mutant proteins were analyzed by Western blotting using anti-FLAG antibody. (B) Protein levels of tim17 mutants after overexpressing under the control of centromeric TEF promoter were analyzed as described earlier. (C) Analysis of protein half-life. Yeast cells expressing C-terminal FLAG-tagged wild-type and mutant Tim17 under an endogenous Tim17 (pRS316 Tim17) background were treated with cycloheximide (50 μg/ml) to inhibit the protein translation; cells were collected at different time intervals. Lysates were prepared and analyzed by Western blotting (AU, arbitrary units). The amounts of different proteins were quantified by densitometry using ImageJ software. (D) Spotting assay. Double mutants from tandem G/AXXXG/A motifs overexpressing Tim17 under the control of centromeric TEF promoter were spotted on medium supplemented with 5-fluoroorotic acid (5-FOA) and incubated at 30°C. Images were captured after 72 h. (E) Growth phenotype analysis. Yeast strains expressing the wild type, tim17_G123L, tim17_G127L (under the control of the endogenous promoter), and tim17_{G123L/G127L-TEF} (under the control of the centromeric TEF promoter) from the TM4 segment were serially diluted, spotted onto the indicated media, and incubated at different temperatures. (F) Protein expression analysis of tim17 mutants. The steady-state protein levels of Tim17 and presequence translocase complex components in TM4 mutants were analyzed by immunodecoration with specific antibodies.

FIG 4

Critical role of Tim17 in regulating both matrix sorting and inner membrane sorting. (A) In vivo precursor accumulation assay. Wild-type and tim17 mutant cells were grown to early log phase and subjected to heat shock at 37°C for 4 h. Cell lysates were prepared and analyzed by Western blotting using Hsp60-specific antibodies; Un, uninduced; In, induced. (B) In vitro import kinetic analysis. Purified mitochondria from wild-type and different mutant yeast strains were subjected to heat shock at 37°C for 30 min to induce phenotype, followed by incubation with saturating amounts of purified cytb2 (1–167)-DHFR at 25°C. The reaction was terminated by the addition of valinomycin at different time intervals; aliquots were subjected to proteinase K treatment. The samples were subsequently analyzed by Western blotting using an anti-DHFR antibody; p, precursor; i and i*, intermediates. (C) The signal intensity of imported intermediate bands obtained from Western blots was quantified by densitometry using ImageJ software for cytb₂ (1–167)-DHFR after normalizing the amounts of precursor imported in the wild type at 20 min as 100% import. (D and E) Mitochondria from wild-type and tim17 mutant cells were subjected to heat shock at 37°C for 30 min to induce phenotype followed by incubation with saturating amounts of purified cytb₂ (1–167)Δ19-DHFR at 25°C. The import reaction was terminated by valinomycin at indicated time intervals, followed by treatment with proteinase K. The samples were subsequently analyzed by Western blotting using an anti-DHFR antibody; p, precursor; i, intermediate (D). The imported intermediate bands were quantified by densitometry using ImageJ software after normalizing the amounts of precursor imported in the wild type at 20 min as 100% import (E).

FIG 5

All the TM regions of Tim17 are essential for Tim23 interaction. (A to E) Purified mitochondria from the wild type and tim17 mutants (TM1 to -4) were subjected to heat shock at 37°C for 30 min to induce phenotype. The mitochondria were lysed with digitonin-containing buffer, and the lysates were subjected to co-IP using anti-FLAG antibody. Samples were separated on SDS-PAGE gels and subjected to Western blot analysis using the indicated specific antibodies to detect the different components of presequence translocase. Twenty-five percent of the total sample served as loading control (Input) against 100% of immunoprecipitated product. The amounts of different immunoprecipitated proteins were quantified by densitometry using ImageJ software.

FIG 6

The integrity of Tim17 transmembrane regions is important for maintaining the architecture of presequence translocase. (A) Analysis of stability of protein complexes using BN-PAGE. The digitonin-solubilized mitochondria from the wild type and tim17 mutants were subjected to BN-PAGE followed by immunoblotting against Tim23 antibodies. The positions of the complexes of the wild type are indicated by arrowheads. Twenty-five percent of the input sample was used as loading control. (B) Co-IP analysis. Tim17 and tim17 TM2 region lethal mutant proteins were C-terminally FLAG-tagged and transformed into a strain harboring endogenous Tim17 (pRS316-Tim17). Mitochondria were isolated and subjected to heat shock at 37°C for 30 min, followed by lysis in digitonin buffer. The lysates were subjected to co-IP using anti-Tim23 antibody. Samples were separated on SDS-PAGE gels and immunoblotted using anti-FLAG antibody. Twenty-five percent of the input was used as loading control.

FIG 7

Cells lacking functional Tim17 protein exhibit a petite-negative phenotype. (A and B) Growth phenotype analysis. Wild-type and mutant yeast cells were grown to mid-log phase at 30°C in liquid YPD medium. Equivalent amounts of cells from each strain were serially diluted and spotted onto YPD medium or YPD medium containing 40 μg/ml ethidium bromide. The plates were incubated at the permissive temperatures for 3 days, and images were captured after 72 h (A). Similarly, the wild type and tim17 conditional mutants were transformed with either ILV5 or ABF2 on a centromeric plasmid under the control of the TEF promoter. The transformed yeast strains were serially diluted and spotted onto YPD medium containing 40 μg/ml ethidium bromide and incubated at the permissive temperatures for 3 days. Images were captured after 72 h (B). (C and D) Quantification of the nuclei number in tim17 mutants. Yeast cells were transformed with mitochondrially targeted mCherry and allowed to grow to mid-log phase at 30°C. The cells were stained with 10 mM SYTO 18 for 20 min, followed by imaging analysis using a Delta Vision Elite fluorescence microscope (GE Healthcare), and the images were analyzed using SoftWoRx 6.1.3 software. The images from mCherry and SYTO 18 were superimposed (merge). The cells in all the panels were imaged at identical exposures to compare the fluorescence intensity levels. Bars, 5 μm. (C). Similarly, wild-type and the indicated tim17 mutant strains were grown to mid-log phase, followed by incubation at the restrictive temperature of 37°C for 0, 4, and 8 h. The mitochondrial nucleoid morphology was examined at each time interval by staining with SYTO 18 to a final concentration of 10 mM, followed by imaging analysis using a Delta Vision Elite fluorescence microscope (D). The cells in all the panels were subjected to identical exposures. Bars, 5 μm.

FIG 8

The functional Tim17 protein is essential for maintaining the membrane polarity across the inner membrane. (A and B) Measurements of inner membrane polarity. Purified mitochondria from wild-type and tim17 mutant cells were stained with JC-1 dye for 15 min at room temperature. The fluorescence emission scan was recorded from 500 to 620 nm with an excitation of 490 nm (A). The mean fluorescence intensity obtained in the spectral analysis was quantified (B). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C and D) Determination of functional mitochondrial mass. The indicated wild-type and tim17 mutant strains were grown to mid-log phase at 30°C, followed by treatment of heat shock at 37°C for 4 h. The cells were subjected to staining with MitoTracker Deep Red-A dye for 20 min and were analyzed by flow cytometer using the BD FACSCanto II analyzer (C). The mean fluorescence intensity obtained in FACS analysis was quantitated to estimate the relative total mass in the wild type and tim17 mutants (D). ***, P ≤ 0.001.

FIG 9

Analysis of ROS levels and mitochondrial morphology in tim17 mutants. (A) Measurement of mitochondrial superoxide levels by MitoSOX staining. WT and mutant yeast strains grown to mid-log phase were given heat shock at 37°C for 8 h followed by staining with MitoSOX and subsequently analyzed by flow cytometry. The relative fluorescence intensity is represented as the fold mean fluorescence intensity (MFI). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) Analysis of total cellular ROS by H₂DCFDA staining. After growing to mid-log phase, WT and mutant yeast strains were given heat shock at 37°C for 8 h, followed by staining with H₂DCFDA, and analyzed by flow cytometry. The relative fluorescence intensity is represented as the fold mean fluorescence intensity (MFI). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) Analysis of mitochondrial morphology. Wild-type and tim17 mutant strains expressing the MTS-mCherry construct were grown to mid-log phase at 30°C. The cells were subjected to heat shock at 37°C and incubated for 4 h to induce the mutant phenotype. Samples were collected and were mounted for imaging analysis using a Delta Vision Elite fluorescence microscope. The images were analyzed using SoftWoRx 6.1.3 software. The cells in all the panels were imaged at identical exposures to compare the fluorescence intensities. Bars, 5 μm.