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. 2016 Jan 19;36(6):1032-47.
doi: 10.1128/MCB.01107-15.

An Outer Mitochondrial Translocase, Tom22, Is Crucial for Inner Mitochondrial Steroidogenic Regulation in Adrenal and Gonadal Tissues

Maheshinie Rajapaksha  1 , Jasmeet Kaur  1 , Manoj Prasad  1 , Kevin J Pawlak  1 , Brendan Marshall  2 , Elizabeth W Perry  2 , Randy M Whittal  3 , Himangshu S Bose  4
Affiliations

An Outer Mitochondrial Translocase, Tom22, Is Crucial for Inner Mitochondrial Steroidogenic Regulation in Adrenal and Gonadal Tissues

Maheshinie Rajapaksha et al. Mol Cell Biol. .

Abstract

After cholesterol is transported into the mitochondria of steroidogenic tissues, the first steroid, pregnenolone, is synthesized in adrenal and gonadal tissues to initiate steroid synthesis by catalyzing the conversion of pregnenolone to progesterone, which is mediated by the inner mitochondrial enzyme 3β-hydroxysteroid dehydrogenase 2 (3βHSD2). We report that the mitochondrial translocase Tom22 is essential for metabolic conversion, as its knockdown by small interfering RNA (siRNA) completely ablated progesterone conversion in both steroidogenic mouse Leydig MA-10 and human adrenal NCI cells. Tom22 forms a 500-kDa complex with mitochondrial proteins associated with 3βHSD2. Although the absence of Tom22 did not inhibit mitochondrial import of cytochrome P450scc (cytochrome P450 side chain cleavage enzyme) and aldosterone synthase, it did inhibit 3βHSD2 expression. Electron microscopy showed that Tom22 is localized at the outer mitochondrial membrane (OMM), while 3βHSD2 is localized at the inner mitochondrial space (IMS), where it interacts through a specific region with Tom22 with its C-terminal amino acids and a small amino acid segment of Tom22 exposed to the IMS. Therefore, Tom22 is a critical regulator of steroidogenesis, and thus, it is essential for mammalian survival.

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Figures

FIG 1
FIG 1
3βHSD2 requires an accessible N terminus for cleavage-independent mitochondrial import. (A) The N-terminal 73-amino-acid and C-terminal 84-amino-acid sequences of 3βHSD2 were compared in seven different species, revealing that they are highly conserved. (B) [35S]methionine-labeled 3βHSD2 was synthesized in a cell-free system, and its folding was analyzed in the presence and absence of different concentrations of nonionic detergents and proteinase K (PK) following import into mitochondria isolated from MA-10 cells. The positions of molecular mass markers (M) are indicated to the left of the gel. (C) Analysis of 35S-3βHSD2 import into the mitochondrial fraction followed by PK digestion of the outer mitochondrial membrane (OMM), intermitochondrial membrane (IMM), and matrix. (D) Mitochondrial import of 3βHSD2, (1-22) COX IV-DHFR, and (1-22) COX IV-3βHSD2. Mito, mitochondria; CFS, cell-free transcription/translation system; Carb, washing with sodium carbonate; S, supernatant; P, pellet. (E) 3βHSD2 determines the N-terminal cleavage pattern. Import of the N-terminal 50 amino acids of COX IV fused with either 3βHSD2 or DHFR into isolated mitochondria. The imported fraction was separated from the unimported fraction, and membrane integration was assessed by sodium carbonate extraction. (F) Overexpression of (1-22) COX IV-DHFR in COS-1 cells in the presence (+) and absence (−) of methotrexate (MTX), followed by identification by Western blotting with a DHFR antibody (ab). The bottom panel shows equivalent loads after staining with a VDAC2 antibody. (G) The metabolic activity of the wild-type 3βHSD2 and chimeric 3βHSD2 cDNA fused with COX IV or 39 amino acids of cytochrome P450scc (SCC) was determined in COS-1 cells expressing the F2 fusion vector. Prog, progesterone; Preg, pregnenolone; Trilo, trilostane. (H) Quantitative measurement of the synthesized progesterone. Data represent means plus standard errors of the means (SEM) (error bars) for three independent experiments performed at three different times.
FIG 2
FIG 2
Role of amino acids 283 to 310 in mitochondrial import of 3βHSD2. (A) Analysis of 35S-Δ283-310 3βHSD2 import into the isolated mitochondria and identification of compartmentalization by centrifugation followed by washing with sodium carbonate. (B) Replacement of amino acids 283 to 310 with the 30-amino-acid StAR pause sequence in 3βHSD2 and analysis of its import into isolated mitochondria as in panel A. (C) Schematic presentation of the different internal amino acids from positions 283 to 310 and also substitution of the sequence of the StAR pause sequence from 31 to 61 amino acids. (D) Analysis of the metabolic activity of the indicated 3βHSD2 deletion mutants after cotransfection of COS-1 cells with the F2 vector. Transfect, transfection. (E) Quantitative measurement of the amount of progesterone synthesized in panel D. Data represent the means plus SEM for three independent experiments performed at three different times. (F) Identification of 3βHSD2 mutant-containing complexes after cell-free synthesized 35S-3βHSD2 was imported into mitochondria isolated from MA-10 cells, solubilized with digitonin, and analyzed by 6 to 16% native gradient PAGE. (G) Compartment-specific localization of WT and mutant 3βHSD2 after overexpression in COS-1 cells followed by staining with a 3βHSD2 antibody. The fractions are mitochondria (Mito) and cytoplasm without lipids (Cyto). The middle and bottom panels show a representative Western blot of the same membrane probed with a mitochondrial VDAC2 antibody and a cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, respectively. (H) Immunoprecipitation of the 3βHSD2 constructs obtained from COS-1 cells in mitochondrial extracts with 3βHSD2 (left) and Tom22 (right) followed by Western blotting with a 3βHSD2 (top) and VDAC2 (Bottom) antibodies. (I to L) Electron microscopic analysis of WT 3βHSD2 (I), 300-310 3βHSD2 (J), 283-289 3βHSD2 (K) and 283-295 3βHSD2 (L). The right panels show an enlarged view of a mitochondrion from the respective left panels. Bars, 0.5 μm (I), 1.0 μm (K), and 2.0 μm (J and L).
FIG 3
FIG 3
Analysis of the 3βHSD2-containing mitochondrial translocase complex. (A and B) 35S-3βHSD2 was imported into isolated mitochondria and then solubilized with various concentrations of lauryl maltoside (LM) after which the complex was analyzed through 4 to 16% native gradient PAGE (A). (Bottom) The equivalent loads of the relative LM-solubilized fractions were analyzed by SDS-PAGE, followed by Western blot staining with 3βHSD2 and VDAC2 antibodies. The complex formed with 0.5% LM was excised from the 1D native PAGE and analyzed through 2nd dimension native gradient PAGE under identical conditions (B). (C) Metabolic conversion assays of mouse adrenal tissues preequilibrated with 0.5% LM overnight and then the coactivator, NAD, and [3H]pregnenolone. The accumulated progesterone was identified by thin-layer chromatography. (D) Quantitative measurement of the progesterone synthesized in panel C. (E) Expression of Tom22 after knockdown with two different concentrations of negative siRNA. (F) Expression and localization of 3βHSD2 within the mitochondria in ΔTom22 MA-10 cells. 3βHSD2 (top) and Tom22 (middle) expression was absent in the outer mitochondrial membrane (O) as well as in the combined inner mitochondrial membrane (IMM) and matrix fraction (I) in ΔTom22 cells. Following N124K and K141D Tom22 expression, 3βHSD2 expression was restored in the IMM fraction, and Tom22 expression was restored in the OMM (O) fraction. (Bottom) VDAC2 expression was equally present in the OMM fraction irrespective of Tom22 knockdown. (G) Western blot analysis showing the expression of mitochondrial matrix-resident aldosterone synthase (AS) and P450scc (SCC) after incubating with different concentrations of Tom22 siRNA. (H) Western blot analysis showing the change in expression of 3βHSD2 after incubating two different concentrations of Tom22 siRNA with the MA-10 cells. (I) Metabolic conversion assays with MA-10 cells incubated with various concentrations of Tom22 siRNA, NAD, and [3H]pregnenolone. The accumulated progesterone was identified by thin-layer chromatography. (Right) Quantitative measurement of the progesterone synthesized in the left panel. (J) Specificity of the 3βHSD2 antibody. Western blot analysis of the indicated amount of 3βHSD1 (AbCam) and 3βHSD2 (our homemade antibody) using antibodies specific for each. Our antibody recognizes only 3βHSD2; however, the commercially available 3βHSD1 antibody recognizes both 3βHSD2 and 3βHSD1. Data presented in panels D and I (right panel) represent the means plus SEM for three independent experiments performed at three different times.
FIG 4
FIG 4
Localization of 3βHSD2 and Tom22 by immune electron microscopy (EM). (A to H) Localization of 3βHSD2 (A and B) or Tom22 (C and D) in a MA-10 cell where most of the 3βHSD2 was localized to the cristae and OMM (B), and Tom22 is localized at the OMM (D). Panels B and D show a higher magnification of a mitochondrion. (E) Visualization of the mitochondrial structure in the absence of any antibody. (F to H) Visualization of the mitochondrion with Tom22 and 3βHSD2 antibodies together in a MA-10 cell. For 3βHSD2, the gold particle was 15 nm (red arrows), whereas the particle size was 55 nm for Tom22 (green arrows). Panels G and H show a higher magnification of a mitochondrion after double labeling. (I to L) COS-1 cells were incubated with antibodies specific for 3βHSD2 (I and J), confirming its absence, and Tom22 expression in mitochondria (K and L). Panel K is a higher-magnification image of a COS-1 cell mitochondrion labeled with Tom22 and 3βHSD2 antibodies. Bars, 1.0 μm (A and F), 0.5 μm (C and K), and is 2.0 μm (I).
FIG 5
FIG 5
3βHSD2 directly interacts with Tom22. (A and B) Coimmunoprecipitation of MA-10 cells incubated with 0.3% LM overnight and then immunoprecipitated with the indicated antibodies followed by Western blotting with 3βHSD2 and Tom22 antibodies. The cross-linked products were separated by 12% SDS-PAGE (A) and 16% SDS-PAGE (B). The bottom panels show Western blotting with VDAC2 and Tom22 or 3βHSD2 antibodies prior to cross-linking. Preimmu, preimmune. (C) In vitro chemical cross-linking of the mitochondria isolated from mouse adrenals permeabilized with LM, cross-linked with various concentrations of BS3, and then separated by 12% SDS-PAGE, followed by Western blotting with Tom22 antibody. A new 60-kDa protein was apparent due to Tom22 and 3βHSD2 interaction. (D) After chemical cross-linking of MA-10 cells with BS3, the cells were lysed with LM, and identification of the chemical cross-linked protein bands was performed by electrophoresis on 16% SDS-polyacrylamide gels followed by Western blotting with 3βHSD2 antibody. The bottom gels in panels C and D show the protein levels prior to cross-linking stained with VDAC2 antibody, confirming that an identical amount of protein was employed in each experiment. (E and F) Localization of the proteins associated with Tom22 and 3βHSD2 through sucrose density gradient after 2 h and 4 h of ultracentrifugation. Each fraction was probed with the indicated antibodies, and the distribution of the proteins was graphed. Data presented are the means ± SEM for three independent experiments. The bottom panels show representative density gradient fractionation and Western blotting with the indicated antibodies.
FIG 6
FIG 6
Role of the C terminus of Tom22 in regulating 3βHSD2 metabolic activity. (A) The indicated Tom22 point mutants were expressed in MA-10 cells after knocking down Tom22 with 60 pmol of siRNA. Western blot analysis was performed using Tom22, 3βHSD2, and VDAC2 antibodies, confirming the equal expression of each. Expression remained unchanged with negative (−) siRNA. (B) Pregnenolone-to-progesterone conversion by ΔTom22 MA-10 cells overexpressing the indicated mutants. (C) Quantitative measurement of the progesterone synthesized. Data represent the means plus SEM for three independent experiments performed at three different times. (D) Co-IP analysis with 3βHSD2 antibodies after Tom22 knockdown and expression of the indicated mutants in MA-10 cells. The blots were stained with 3βHSD2 and Tom22 antibodies independently. The bottom panel shows VDAC2 expression, showing the same amount of cells were used for each experiment. (E to I) Electron microscopy of Tom22 mutants (F to I) in ΔTom22 MA-10 cells. (E) Typical pattern of an enlarged view of mitochondrion stained with 3βHSD2 and Tom22 antibodies. (F) Expression pattern of Tom22 (bottom panel), and the top panel shows its typical localization in a mitochondrion. The small gold particles (15 nm, red arrowheads) are 3βHSD2, and the larger 55-nm particles are Tom22 (green arrows). (G) Tom22 expression is absent in ΔTom22 MA-10 cells, and the top panel shows only one mitochondrion with minimal Tom22. (F and G) Expression of Tom22 in wild-type (F) and siRNA knockdown (G) MA-10 cells. (H and I) Bottom panels show expression of N124K (H) and K141D (I) Tom22 in ΔTom22 MA-10 cells. The top panels show an enlarged mitochondrion from the bottom panel. Bars, 2.0 μm (F) and 1.0 μm (G to I). (J) RT-PCR amplification of cDNA from ΔTom22 MA-10 cells with different 3βHSD2-nested primers showing amplification of 747-bp, 394-bp, and 600-bp cDNA. The human 3βHSD2 plasmid DNA was the control for the PCR, and WT MA-10 cDNA was used as internal control for the siRNA knockdown. The positions of DNA markers (in base pairs) are shown to the right of the gel.
FIG 7
FIG 7
The C terminus of 3βHSD2 is required for interaction with Tom22. (A and B) Metabolic activity of COS-1 cells transfected with the indicated deletion mutants and F2. (Bottom) Quantitative measurement of the amount of progesterone synthesized after three independent transfections performed at three different times. (C) Western blot analysis of COS-1 cells transfected with the indicated C-terminal deletion mutants of 3βHSD2. (D) Coimmunoprecipitation of the lysates isolated from panel C with Tom22 antibodies followed by blotting with 3βHSD2 antibodies. (Bottom) Tom22 and VDAC2 antibodies were employed to provide the evidence of identical loading. (E) Outer (O) and inner (I) mitochondrial fractionation and localization of the WT, C-3, C-7, and C-10 3βHSD2 after overexpression in COS-1 cells and staining with a 3βHSD2 antibody. The bottom panel was stained with a VDAC2 antibody, confirming that an equivalent amount of total protein was present in each lane.
FIG 8
FIG 8
Localization of WT 3βHSD2, C-terminal deletion mutants of 3βHSD2 and Tom22 by immune electron microscopy (EM). EM was undertaken by double labeling with independent antibodies together after overexpression of WT 3βHSD2 and its C-terminal deletion mutants in COS-1 cells. (A) Colocalization of 3βHSD2 and Tom22. (B to E) Localization of C-terminal truncated 3βHSD2 and Tom22. The right panels show an enlarged view of a mitochondrion or a few mitochondria closely associated from the respective left panel. The small 15-nm gold particles (red arrows) show 3βHSD2, and the big 55-nm gold particles indicate Tom22 (green arrows). For 3βHSD2, the gold particle was 15 nm (red arrow); the particle size was 55 nm for Tom22 (green arrow).
FIG 9
FIG 9
Schematic presentation of 3βHSD2 translocation through the mitochondrial Tom40 import channel and association with the IMS-exposed Tom22. In the first step, newly synthesized 3βHSD2 is processed for mitochondrial import through the Tom40 import channel. The translocases are indicated in their appropriate position. In the second step, 3βHSD2 moves closer to the mitochondrial import pore and is imported through the region from amino acids 283 to 310 as a signal sequence (blue). In the final step, following import into the IMS, the 283-310 amino acid region is associated with the IMS-exposed region of Tom22 for participating in metabolic activity. IMM, inner mitochondrial membrane; IMS, intermembrane space; OMM, outer mitochondrial membrane.
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