This study clearly shows that transfection of Y1-BS1 cells (a mouse adrenal cell line) with rat SR-BI using a calcium phosphate precipitation technique results in cells with a dramatically altered cell surface. One version of this disrupted surface can be described as an explosion of SR-BI stained vesicles -where the original plasma membrane (including caveoli, clathrin coated pits, typical microvillar structures) no longer exists, but appears to have become part of the vesicular mass. However, in most of the transfected cells one also sees the development of multiple and quite elaborate SR-BI stained double membraned channel structures at the cell surface, which in their width, their intertwining curving nature, and their general architecture resemble the complex double membraned structures seen associated with the microvillar compartment of hormone-stimulated adrenocortical or ovarian cells in tissues of the normal rat. It is clear that the morphological plasma membrane changes we see are always associated with SR-BI (as they are in intact sections of steroidogenic tissue), and in the case of the Y1-BS1 cell preparations, only cells showing accumulations of SR-BI, show any of the physical changes noted.
In addition to these cell surface related changes, SR-BI transfected cells may also show accumulations of SR-BI intracellularly, often associated with non-identifiable vacuoles or masses found deep within the cell. Along the edges of these masses, patches of SR-BI accumulation can occasionally be identified, and, no matter how big or small the SR-BI mass, it is always associated with double membraned structures – some circular, some long, but in every way identical morphologically to the double membraned channel structures associated with the cell surface. While it is possible that these intracellularly located structures are, in fact, connected to the cell surface (at a point not visible in our sections), it seems possible that these deep sites are where SR-BI has accumulated, but has not been appropriately transported to the cell surface. Perhaps, in such cells, the production of SR-BI is so large that transport and utilization of the protein at the surface does not have sufficient time (or sufficient delivery proteins or equipment) to occur. And, like in SF9 insect cells infected with SR-BII (Reaven & Azhar, unpublished observations), the stalled delivery system deposits the protein at some site, and with the help of available cell machinery, the double membraned channels meant for the surface are actually produced in situ.
How SR-BI is transported through the cell is not yet clear. The multiple glycosylation sites of SR-BI  strongly suggest that the nascent protein must pass through trans Golgi membranes, but remarkably little SR-BI is visible in, or around, the Golgi areas, despite the use of multiple antibody types (anti SR-BI against the C-terminal or extracellular domain (ECD) or V5 or cMYc tagged SR-BI). Indeed, the most heavily transfected cells do not seem to have easily identifiable Golgi areas at all, and one wonders if transport of the newly formed protein has exhausted the Golgi membranes-which then are distributed throughout the cell and perhaps form the basis for some of the intracellular sites of SR-BI which we do see. Likewise, the existing endoplasmic reticulum is not heavily labeled for SR-BI, though in occasional cells one can find patches of wavy ER-like membranes (some even with attached ribosomes) which appear to stain lightly for SR-BI. Perhaps passage of the nascent protein through these traditional membranes is too rapid a process to show up dramatically. Or, as is always possible, the antigenic sites of SR-BI may be masked at these early stages, and it is not until the protein later dimerizes in situ, or at the cell surface, that the protein becomes available for immunostaining.
What remains clear is that cellular sites where SR-BI is prominent (e.g. surface membrane + surface or intracellular craters) show a high degree of SR-BI dimerization. Approximately 40% of the gold particles found in such areas are in close contact with each other, and this remains true whether the immuno-gold represents staining of two SR-BI V5 molecules, two SR-BI cMyc molecules, or a combination of the two different tags (therefore a dimer composed of SR-BI-V5+SR-BI cMyc). Indeed, the double tagged dimer re-enforces the idea that SR-BI, whatever its identification tag, migrates to the same cell region, and is intimately involved in the construction of the double membraned channels which result.
Finally, to what extent do the attributes of an exquisitely complex cell surface of double membraned channels composed of highly dimerized molecules of SR-BI lead to heightened cell function? We suggest that cells with coils of double membraned channels containing dimerized SR-BI are capable of taking up vast numbers of HDL particles [13, 14] in any given preparation. Such cells with their substantially increased cell surface and heightened ability to attract and trap HDL will also deliver increased CE mass to the cell interior as substrate for increased steroid production.