Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: implications for the mechanism of StAR action

Abstract

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone biosynthesis, presumably by facilitating the delivery of cholesterol to P450scc in the inner mitochondrial membranes. StAR is synthesized as a 37-kDa preprotein that is processed to a 30-kDa mature form by cleavage of an N-terminal mitochondrial import sequence. To identify structural features required for StAR biological activity, we mutated the human StAR cDNA, including the deletion of N- and C-terminal sequences, and examined the ability of the mutants to promote steroidogenesis and enter the mitochondria of transfected COS-1 cells. Deletion of up to 62 residues from the N terminus (N-62) did not significantly affect steroidogenesis-enhancing activity. The N-terminal deletion mutants were associated with mitochondria-enriched fractions, but import and processing were progressively impaired with increasing length of the deletion. Immunogold electron microscopy and in vitro import assays showed that the active N-62 mutant was not imported into the mitochondria. Removal of the 28 C-terminal amino acids (C-28) inactivated StAR. Deletion of the C-terminal 10 amino acids (C-10) reduced steroidogenic activity by 53%, while truncation of the last 4 amino acids had no effect. The C-28 mutant StAR was not efficiently imported into mitochondria or processed, whereas some of the C-10 mutant was processed, indicating that import had occurred. We conclude that in the COS-1 cell system used, StAR does not need to enter into mitochondria to stimulate steroidogenesis and that residues in the C terminus are essential for steroidogenesis-enhancing activity. These findings imply that StAR acts via C-terminal domains on the outside of the mitochondria.

Figure 1

Deletions of the N terminus of StAR do not significantly impair its steroidogenic activity. The indicated constructs were expressed in COS-1 cells with the cholesterol side-chain cleavage system. Transfected cells were incubated without (solid bars) and with 22(R)-hydroxycholesterol (open bars) as described in the text. Pregnenolone secretion, determined by radioimmunoassay, was expressed as nanograms per dish per 48 h for a representative experiment (X ± SD, n = 3 triplicate culture dishes) (A) or as a percentage of that secreted by cells transfected with the wild-type StAR (B) where values are means ± SEM from three separate experiments with triplicate cultures in each treatment. (B) The mean secretion of pregnenolone by COS-1 cells transfected with wild-type StAR using endogenous cholesterol was 670 ng per dish per 48 h. The mean pregnenolone secretion by these cells in the presence of 22(R)-hydroxycholesterol was 1811 ng per dish per 48 h.

Figure 2

(A) Western blot analysis of N-19 and N-28 mutants expressed in COS-1 cells. Mitochondria-enriched fractions were prepared from COS-1 cells transfected with the indicated constructs expressing wild-type or mutant StAR proteins. (B and C) N-48 and N-62 StAR mutants are not imported into mitochondria or processed. Mitochondria-enriched subcellular fractions were prepared from COS-1 cells transfected with plasmids for wild-type StAR or the N-48 and N-62 mutants. One aliquot was incubated in buffer, the other in proteinase K; in some cases (C), an aliquot was incubated with 0.05% Triton X-100, before SDS/PAGE and Western blotting for StAR protein.

Figure 3

Immunogold electron microscopy detects wild-type StAR in the mitochondrial matrix, while the N-62 mutant is primarily localized to the mitochondrial outer membranes. (A) Mitochondrion from COS-1 cell transfected with empty plasmid; (B) mitochondrion from COS-1 cell transfected with wild-type StAR; and (C) mitochondrion from COS-1 cell transfected with N-62 mutant. (D) Mitochondrion from COS-1 cell transfected with wild-type StAR; (E and F) mitochondria from COS-1 cells transfected with N-62 mutant. (A–C) Fixation for immunoelectron microscopy; (D–F) fixation for routine electron microscopy with antigen recovery. Arrows indicate 10-nm gold particles. (Bar = 0.25 μm.)

Figure 4

The wild-type StAR preprotein is imported and processed by mouse liver mitochondria, but the N-62 mutant is not. ³⁵S-labeled wild-type StAR and N-62 mutant were incubated with mouse mitochondria as described. Imported and processed wild-type StAR is present in mitochondria following proteinase K digestion in the absence of Triton X-100.

Figure 5

Truncations of the C terminus of StAR abrogate steroidogenesis-enhancing activity. COS-1 cells were transfected with plasmids expressing wild-type StAR and the indicated mutants with the cholesterol side-chain cleavage system. The transfected cells were incubated without (solid bars) or with 22(R)-hydroxycholesterol (open bars), and pregnenolone secretion was determined by radioimmunoassay. Values presented are nanograms per dish per 48 h (means ± SD, n = 3 dishes per treatment) from representative experiments (A and B) or relative to the pregnenolone secretion by cells transfected with the wild-type StAR as means ± SEM (n = 3 separate experiments with three dishes per treatment in each experiment) (C). Pregnenolone secretion by cells transfected with wild-type StAR averaged 439 ng per dish per 48 h from endogenous cholesterol and 850 ng per dish per 48 h in the presence of 22(R)-hydroxycholesterol.

Figure 6

Western blot analysis of C-terminal truncation mutants. COS-1 cells were transfected with plasmids expressing wild-type StAR or the indicated mutants. Mitochondria-enriched subcellular fractions were subjected to Western blotting for StAR protein.

Figure 7

The C-28 mutant is not efficiently imported into mitochondria or processed. ³⁵S-labeled wild-type StAR and C-28 mutant were analyzed in the in vitro import assay. Wild-type StAR is imported and processed yielding proteinase K resistant mature protein. The C-28 mutant is not imported.