Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins.

Steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein that enhances steroid synthesis by facilitating the transfer of cholesterol to the inner membranes of mitochondria in hormonally regulated steroidogenic cells. It is currently assumed that StAR activity commences before or during StAR import into the mitochondrial matrix. The present study was designed to demonstrate that, once imported and becoming physiologically irrelevant, exhaustive accumulation of StAR must be limited by a rapid degradation of the protein to prevent potential damage to the organelles. The use of uncouplers and manipulation of the interior mitochondrial pH in hormone-induced ovarian granulosa cells and StAR-expressing COS cells suggests that StAR degradation is biphasic and involves two classes of proteases. During phase I, which normally lasts for the first approximately 2 h following import, StAR is rapidly degraded by a protease, or proteases, that can be arrested by a nonclassical action of proteasome inhibitors such as MG132. StAR molecules that evade phase I are subjected to a second class of protease(s), which is slower and MG132 resistant. A third proteolytic entity was revealed in studies with C-28 StAR, a loss-of-function mutant of StAR. Upon initiation of its import, C-28 StAR dissipates the inner membrane potential and causes swelling of the mitochondria. Degradation of C-28 StAR, probably by an intermembrane space protease, is extremely rapid and MG132 insensitive. Collectively, this study defines StAR as the first naturally occurring mitochondrial protein that can serve as a substrate to probe multiple proteolytic activities in mammalian mitochondria.

The roles of circulating high-density lipoproteins and trophic hormones in the phenotype of knockout mice lacking the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory protein (StAR) is essential for the regulated production of steroid hormones, mediating the translocation of intracellular cholesterol to the inner mitochondrial membrane where steroidogenesis begins. Steroidogenic cells lacking StAR have impaired steroidogenesis and progressively accumulate lipid, ultimately causing cytopathic changes and deterioration of steroidogenic capacity. Developmental studies of StAR knockout (KO) mice have correlated gonadal lipid deposits with puberty, suggesting that trophic hormones contribute to this lipid accumulation. To delineate the role of gonadotropins in this process, we examined double mutant mice deficient in both StAR and gonadotropins [StAR KO/hpg (hypogonadal)]. Lipid accumulation was ameliorated considerably in StAR KO/hpg mice but was restored by treatment with exogenous gonadotropins, directly linking trophic hormones with gonadal lipid accumulation. To define the relative roles of exogenous vs. endogenous cholesterol in the lipid accumulation, we also examined mice lacking both StAR and apolipoprotein A-I (StAR KO/Apo A-I KO). Steroidogenic tissues of StAR KO/Apo A-I KO mice had markedly decreased lipid deposits, supporting the predominant role of high-density lipoprotein-derived cholesterol in the lipid accumulation caused by StAR deficiency. Finally, we used electron microscopy to compare mitochondrial ultrastructure in StAR KO and cholesterol side-chain cleavage enzyme (Cyp11a1) KO mice; despite comparable lipid accumulation within adrenocortical cells, the effects of StAR deficiency and Cyp11a1 deficiency on mitochondrial ultrastructure were markedly different. These findings extend our understanding of steroidogenic cell dysfunction in StAR KO mice and highlight key roles of trophic hormones and high-density lipoprotein-derived cholesterol in lipid deposits within StAR-deficient steroidogenic cells.

The life cycle of the steroidogenic acute regulatory (StAR) protein: from transcription through proteolysis.

The Steroidogenic Acute Regulatory (StAR) protein is a mitochondrial protein required for the transport of cholesterol substrate to the P450scc enzyme located in the inner mitochondrial membranes of steroid producing cells. This study suggests that the acute regulation of the rodent StAR gene in the ovary is mediated by two factors, C/EBPbeta and GATA-4. Once translated, the StAR precursor protein is either imported into the mitochondria, or it is rapidly degraded in the cytosol. We predicted that in order to perpetuate StAR activity cycles, imported StAR should turn over rapidly to avoid a potentially harmful accumulation of the protein in sub-mitochondrial compartments. Pulse-chase experiments in metabolically labeled cells showed that: (a) the turnover rate of mature mitochondrial StAR protein (30 kDa) is much faster (t(1/2) = 4-5 h) than that of other mitochondrial proteins; (b) dissipation of the inner membrane potential (-delta psi) by carbonyl cyanide m-chlorophenylhydrazone (mCCCP) accelerates the mitochondrial degradation of StAR; (c) unexpectedly, the mitochondrial degradation of StAR is inhibited by MG132 and lactacystin, but not by epoxomicin. Furthermore, StAR degradation becomes inhibitor-resistant two hours after import. Therefore, these studies suggest a bi-phasic route of StAR turnover in the mitochondria. Shortly after import, StAR is degraded by inhibitor-sensitive protease(s) (phase I), whereas at later times, StAR turnover proceeds to completion through an MG132-resistant proteolytic activity (phase II). Collectively, this study defines StAR as a unique protein that can authentically be used to probe multiple proteolytic activities in mammalian mitochondria.

Regulation of light-dependent Gqalpha translocation and morphological changes in fly photoreceptors.

Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. Little is known, however, about the regulation of Galpha subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqalpha to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqalpha affect these processes. Epistatic analysis showed that Gqalpha is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqbeta is essential for targeting of Gqalpha to the membrane and suggested that Gqbeta is also needed for efficient activation of Gqalpha by rhodopsin. Our results support the 'two-signal model' hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity-dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.