Abstract
The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3′- and 5′-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles.
Highlights
Steroidogenic acute regulatory protein (StAR) functions as a key determinant of steroidogenesis by transferring cholesterol from the outer mitochondrial membrane (OMM) to Cyp11a1 in the inner mitochondrial membrane (IMM) [1,2,3,4]
Previous work to elucidate the mechanism of mitochondrial cholesterol transfer has largely focused on the COS-1 model, in which StAR cholesterol-binding domain (CBD) is transfected with a functioning mitochondrial Cyp11a1 system [11, 52]
The StAR CBD is sufficient to obtain maximum cholesterol transfer, including when this domain anchored to the OMM
Summary
Steroidogenic acute regulatory protein (StAR) functions as a key determinant of steroidogenesis by transferring cholesterol from the outer mitochondrial membrane (OMM) to Cyp11a1 in the inner mitochondrial membrane (IMM) [1,2,3,4]. Inhibition with cycloheximide (CHX) halts adrenocorticotropic hormone (ACTH)-stimulated steroidogenesis within 5 min, while accumulating cholesterol in the OMM remains inaccessible to IMM Cyp11a1 [24,25,26] This intermembrane cholesterol barrier in the adrenal mitochondria caused by CHX treatment is readily breached by mild mitochondrial disruption, including the elevation of Ca2+. Such artificial transfer removes succinate-initiated NNT support for this CHX-sensitive Cyp11a1 activity [6]. Br-cAMP rapidly stimulates Znf36l1/ Tis11b, which binds to specific dual AU-rich elements in the extended 3′UTR This homodimer complex recruits ribonucleases to selectively degrade the 3.5-kb StAR mRNA. The work presented here provides evidence that mRNA generation, mitochondrial positioning, translation, and N-terminal targeting of new StAR proteins may be coordinated to match organizational steps involving cholesterol transfer across the mitochondrial membranes, consistent with an integrated CHXsensitive mechanism
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