Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors.
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.
Granot
Zvi
Z
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Kobiler
Oren
O
Melamed-Book
Naomi
N
Eimerl
Sarah
S
Bahat
Assaf
A
Lu
Bin
B
Braun
Sergei
S
Maurizi
Michael R
MR
Suzuki
Carolyn K
CK
Oppenheim
Amos B
AB
Orly
Joseph
J
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D012739:Gonadal Steroid Hormones / Q000096:biosynthesis; D006107:Granulosa Cells / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D049070:Protease La / Q000502:physiology*; D061988:Proteasome Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D000098682:Steroidogenic Acute Regulatory Protein
D012739:Gonadal Steroid Hormones; D010750:Phosphoproteins; D061988:Proteasome Inhibitors; D000098682:Steroidogenic Acute Regulatory Protein; D000255:Adenosine Triphosphate; D049070:Protease La