Steroidogenic Acute Regulatory Protein Binds Cholesterol and Modulates Mitochondrial Membrane Sterol Domain Dynamics

Abstract

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K d values near 32 nM), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R 2/3 ‫؍‬ 33 Å) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.

Crystal structure of the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-related lipid transfer domain

“…Initial calculations indicated that the StarD4 tunnel volume is 1,424 Ϯ 294 Å 3 (a similar value of 1,820 Ϯ 481 Å 3 was observed with MLN64). These results compare favorably with the previously reported estimate of 1,900 Å 3 for MLN64 (8), which is compatible with the binding of two molecules of cholesterol (28). Visual inspection of the two tunnels, however, suggests that only one cholesterol molecule can be accommodated by either protein.…”

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…However, SCP-2 overexpression still reduced the early exchange of sterol as seen by the 6-fold reduction in initial rate by shifting most of the sterol into the nonexchangeable pool (173). In part, this might explain the large pool of non-exchangeable sterol observed in exchange assays using mitochondrial membrane vesicles isolated from steroidogenic MA-10 Leydig cells (143).…”

“…DHE has been used to probe the structural (polarization, limiting anisotropy, order), dynamic (lifetime, rotational rate), and kinetic (exchange) environment of cholesterol in plasma membranes (6,7,54,55,135,(138)(139)(140)(141), microsomes (6,7), mitochondria (6,7,142,143), and lysosomes (8,129). DHE has revealed several important structural and dynamic aspects of the cholesterol microenvironment in biological membranes.…”

Impact of Aging on Steroid Hormone Biosynthesis and Secretion

“…StAR is synthesized as a short-lived cytoplasmic 37-kDa protein with a mitochondrial leader peptide that is cleaved upon mitochondrial import to yield the long-lived intramitochondrial 30-kDa form [58]. It is now wellestablished that StAR functions as a sterol transfer protein [70], binds cholesterol [71,72], mediates the acute steroidogenic response by moving cholesterol from the outer to the inner mitochondrial membrane [73], acts on the outer mitochondrial membrane [73][74][75], and requires the structural change previously described as a pH-dependent molten globule [76]. StAR is also a prototype of a family of proteins that contain START (StAR-related lipid transfer) domains (StarD proteins) [77], of which StarD3/MLN64, StarD4, StarD5 and StarD6 exhibit steroidogenic potential [78,79].…”