Turnover of Mitochondrial Steroidogenic Acute Regulatory (StAR) Protein by Lon Protease: The Unexpected Effect of Proteasome Inhibitors

Abstract

Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein promoting transfer of cholesterol into steroid making mitochondria in specialized cells of the adrenal cortex and gonads. Our previous work has demonstrated that StAR is rapidly degraded upon import into the mitochondrial matrix. To identify the protease(s) responsible for this rapid turnover, murine StAR was expressed in wild-type Escherichia coli or in mutant strains lacking one of the four ATP-dependent proteolytic systems, three of which are conserved in mammalian mitochondria-ClpP, FtsH, and Lon. StAR was rapidly degraded in wild-type bacteria and stabilized only in lon (-)mutants; in such cells, StAR turnover was fully restored upon coexpression of human mitochondrial Lon. In mammalian cells, the rate of StAR turnover was proportional to the cell content of Lon protease after expression of a Lon-targeted small interfering RNA, or overexpression of the protein. In vitro assays using purified proteins showed that Lon-mediated degradation of StAR was ATP-dependent and blocked by the proteasome inhibitors MG132 (IC(50) = 20 microm) and clasto-lactacystin beta-lactone (cLbetaL, IC(50) = 3 microm); by contrast, epoxomicin, representing a different class of proteasome inhibitors, had no effect. Such inhibition is consistent with results in cultured rat ovarian granulosa cells demonstrating that degradation of StAR in the mitochondrial matrix is blocked by MG132 and cLbetaL but not by epoxomicin. Both inhibitors also blocked Lon-mediated cleavage of the model substrate fluorescein isothiocyanate-casein. Taken together, our former studies and the present results suggest that Lon is the primary ATP-dependent protease responsible for StAR turnover in mitochondria of steroidogenic cells.

Adaptor-mediated Lon proteolysis restrictsBacillus subtilishyperflagellation

“…Eukaryotic Lon (37% identical to LonA of B. subtilis) (SI Appendix, Fig. S7) is involved in both the general degradation of misfolded proteins and in the specific remodeling of the mitochondrial proteins cytochrome C oxidase COX4-1, transcription factor A (TFAM), and the cholesterol regulator StAR (47)(48)(49)(50). It has been suggested that Lon could be a viable anticancer drug target as drugs that inhibit Lon activity lead to cancer cell apoptosis in vitro either due to uncontrolled accumulation of misfolded proteins in general or due to the failure to control a specific regulator (48,51,52).…”

Commitment of 1-Methyl-4-phenylpyrinidinium Ion-induced Neuronal Cell Death by Proteasome-mediated Degradation of p35 Cyclin-dependent Kinase 5 Activator

“…Mitochondrial proteins would similarly fail to be imported into the mitochondria. If mitochondrial proteins are not imported into the mitochondria properly, they are degraded rapidly by proteasomes (48). For example, preornithine transcarbamylase is degraded by proteasomes in the cytosol when its import is inhibited by oxidative stress (38).…”

“…Ubiquitin-independent proteasomal degradation has been demonstrated for several mitochondrial proteins. For example, steroidogenic acute regulatory protein (StAR) is proteolysed independently of ubiquitin in the cytoplasm by proteasomes (48). Preornithine transcarbamylase is degraded in the cytoplasm by proteasomes when mitochondrial import is inhibited by Paraquat (38), but its ubiquitination was not detected despite extensive experimental efforts.…”

Quality Control of Mitochondrial Proteostasis

“…Perhaps such substrates are more prone to misfolding or to damage in conditions of mitochondrial stress. In mammalian mitochondria, LON has been shown to degrade aconitase, an Fe/S protein, the steroidogenic acute regulatory protein (StAR), and the mitochondrial transcription factor A (TFAM) Granot et al 2007;Matsushima et al 2010). LON appears to play a role in cellular aging as in aged mice, the levels of functional LON decline, concomitant with an increase in oxidatively damaged mitochondrial proteins and increased mitochondrial dysfunction (Bota et al , 2005.…”