Inner Mitochondrial Translocase Tim50 Interacts with 3β-Hydroxysteroid Dehydrogenase Type 2 to Regulate Adrenal and Gonadal Steroidogenesis

Pawlak, Kevin J.; Prasad, Manoj; Thomas, James L.; Whittal, Randy M.; Bose, Himangshu S.

doi:10.1074/jbc.m111.290031

Cited by 37 publications

(78 citation statements)

References 53 publications

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“…Mutant Tom22 activity was measured after knocking down Tom22 by siRNA for 24 h followed by transfection of the mutant Tom22 cDNA using Lipofectamine. siRNA transfection was done with 60 pmol of siRNA using Oligofectamine as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

“…The cross-linked cells were collected by centrifugation at 3,000 rpm and resuspended in 10 mM HEPES, pH 7.4. Organelle fractionation was performed following a standard procedure previously described (25,28).…”

Section: Methodsmentioning

confidence: 99%

“…We used the same sequence for knocking down Tom22 as previously described (25). To knockdown VDAC2 expression, MA-10 or COS-1 cells were incubated with 30 or 60 pmol siRNA (sense, 5=-GGUUAGCCUACUCUCUAAUT T-3=; antisense, 5=-AUUAGAGAGUAGGCUAACCTT-3=) for 24, 36, and 48 h after which target protein expression was determined by Western blotting with specific antibodies.…”

Section: Methodsmentioning

confidence: 99%

“…After the solvent was evaporated under N 2 , the extract was resuspended in 100 l methanol (MeOH), and radioactive counts were determined. The endogenous progesterone activity was determined (25,32), and the steroids were characterized by gas chromatography and mass spectrometry (33).…”

Section: Methodsmentioning

confidence: 99%

“…6A). Since Tom22 expression is associated with 3␤HSD2, we next examined pregnenolone-toprogesterone conversion following expression of the C-terminal Tom22 mutants (25). Whereas WT MA-10 cells synthesized 97 ng/ml of progesterone, the N124K mutant synthesized 61 ng/ml, which is about 60% (Fig.…”

Section: ␤Hsd2 Amino and Carboxyl Termini Are Strongly Conservedmentioning

confidence: 99%

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

Rajapaksha

Kaur

Prasad

et al. 2016

Molecular and Cellular Biology

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dAfter 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 3␤-hydroxysteroid dehydrogenase 2 (3␤HSD2). 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 3␤HSD2. 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 3␤HSD2 expression. Electron microscopy showed that Tom22 is localized at the outer mitochondrial membrane (OMM), while 3␤HSD2 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. Mitochondrial function is not limited to oxidative phosphorylation, as mitochondria are the site of steroid synthesis in specialized cells. Most mitochondrial proteins are expressed by nuclear genes with the mitochondrial targeting information in the mature protein. For example, some inner mitochondrial matrix (IMM) proteins have an internal, positively charged "presequence"-like signal, often preceded by a hydrophobic sequence (1), which leads to the arrest of translocation in the IMM and the subsequent lateral release of the protein into the lipid phase of the membrane (2). Import and subsequent intramitochondrial sorting of mitochondrial proteins are mediated by the mitochondrial membrane protein translocases. The translocase complexes in the outer mitochondrial membrane (OMM) are not tightly linked with those of the IMM; yet, they dynamically cooperate to achieve protein delivery to each mitochondrial subcompartment. To understand the mechanism of protein transport by the mitochondrial import/sorting system, it is essential to monitor and analyze the dynamic interactions among the constituents of the system. Steroidogenic cells do not store steroid hormone; therefore, an immediate steroidogenic response requires rapid synthesis of new steroid. Cholesterol mitochondrial transport initiates inner mitochondrial metabolic activity in steroidogenic cells within adrenal and gonadal tissues as well as the brain. Conversion of the first steroid, pregnenolone, to progesterone is catalyzed by 3␤-hydroxysteroid dehydrogenase 2 (3␤HSD2). Due to its central role in steroidogenesis, changes in 3␤HSD2 activity can have a wide range of effects, including hypertension, salt wasting crisis, and impaired sexual development (3-7). We have also identified p...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: ␤Hsd2 Amino and Carboxyl Termini Are Strongly Conservedmentioning

confidence: 99%

See 3 more Smart Citations

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

Rajapaksha

Kaur

Prasad

et al. 2016

Molecular and Cellular Biology

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Cortical adrenal mitochondrial morphology changes in functional state: new insights

et al. 2012

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We compared changes in the morphology of mitochondrial cristae with those in the blood and adrenal content of steroid hormones after the stimulation or inhibition of steroidogenesis. Rats were treated with adrenocorticotrophic hormone or angiotensin II to elicit steroidogenesis and with dexamethasone to inhibit it. Blood and adrenal glands were collected after several time intervals for measurements of steroids and their main intermediates. In the zona fasciculata, mitochondrial ultrastructure was investigated by high resolution scanning electron microscopy. We found that the morphometric data correlated well with the measurements of hyper- or hypo-activity of steroidogenesis over short periods of time (4 h) but not over longer observation times. A peculiar finding was that, contrary to previous reports, 11-deoxycortisol was present in adult rat adrenal tissue.

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3β-Hydroxysteroid Dehydrogenase Deficiency

Morel

Roucher

Plotton

et al. 2014

Genetic Steroid Disorders

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Inner Mitochondrial Translocase Tim50 Interacts with 3β-Hydroxysteroid Dehydrogenase Type 2 to Regulate Adrenal and Gonadal Steroidogenesis

Cited by 37 publications

References 53 publications

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

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

Cortical adrenal mitochondrial morphology changes in functional state: new insights

3β-Hydroxysteroid Dehydrogenase Deficiency

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