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Cholesterol transporter caveolin-1 transits the lipid bilayer during intracellular cycling¹

MIRKO J. ROBENEK^*, KARIN SCHLATTMANN, KLAUS-PETER ZIMMER^*, GABRIELE PLENZ, DAVID TROYER and HORST ROBENEK^,2

Department of Cell Biology and Ultrastructure Research, Institute for Arteriosclerosis Research,
^* University Children’s Hospital, and
Department of Cardiology and Angiology and Department of Thoracic and Cardiovascular Surgery, University of Münster, Germany

²Correspondence: Department of Cell Biology and Ultrastructure Research, Institute for Arteriosclerosis Research at the University of Münster, Domagkstrasse 3, 48149 Münster, Germany. E-mail: robenek{at}uni-muenster.de

SPECIFIC AIMS

At the cell surface, caveolin-1, a ubiquitous, caveolar protein known to cycle between the plasma membrane and membranes of the intracellular compartments, is incorporated into one membrane leaflet only, as its C- and N-terminals project from the plasma membrane into the cytosol, and its lipophilic moiety incompletely spans the lipid bilayer. One premise of all current mechanistic models of caveolin-1 cycling is that caveolin-1 is inserted into the cytoplasmic leaflets in the plasma membrane and in membranes of the intracellular compartments, this configuration being inherently expedient for trafficking caveolin-1 from one membrane to another via the endocytotic pathway. As this assumed disposition of caveolin in cell membranes has never been confirmed or challenged, we aimed to determine in which leaflets of the membranes caveolin-1 does in fact reside.

PRINCIPAL FINDINGS

1. Caveolin-1 resides in the protoplasmic (P)-face (cytoplasmic leaflet) of the plasma membrane
We used freeze-fracture immunolabeling to determine in which cellular compartments caveolin-1 occurs in smooth muscle cells. In this procedure, frozen cells are fractured, and the exposed surfaces are replicated by shadowing with platinum and carbon under vacuum. The replicas are washed with sodium dodecyl sulfate (SDS) to flush away cellular residues and retain only those molecules that adhere directly to the metal of the replicas. We labeled caveolin-1 remaining on the replicas using a monoclonal antibody against caveolin-1 followed by a gold conjugate; viewed in the electron microscope, gold particles mark caveolin-1 in the replicas. Freeze-fracturing always splits membranes into their two constituent leaflets, as the plane of cleavage runs preferentially between the tails of the phospholipids in the bilayer. Whether the cytoplasmic (P-face) or the exoplasmic leaflet (E-face) of the membrane is exposed and hence, into which leaflet caveolin-1 is integrated can be unequivocally ascertained from the topography of the replica. Various membrane integral proteins appear as so-called intramembrane particles.

In surface view, caveolae in the plasma membrane appear as small dimples in the P-face and bumps in the E-face of varying depth and height and of roughly equal size (Fig. 1 A–C). Caveolae are often concentrated in patches, but caveolae-poor regions of the membrane are also present, even in cells with elsewhere-abundant caveolae.

View larger version (174K): [in this window] [in a new window]   Figure 1. Caveolin-1 is present only on the P-face (cytoplasmic leaflet) of the plasma membrane. A) Exposed P- and E-face of the plasma membrane. Caveolae (cav) appear as bumps in the E-face and dimples in the P-face. Colloidal gold particles label caveolin-1 on the P-face at caveolae and in undifferentiated areas of the membrane (arrowheads). The E-face is completely unlabeled in the same fracture surface. B) P-face of the plasma membrane with caveolin-1 labeling mainly at the rims of deep caveolae (arrowheads). C) P-face of the plasma membrane with weaker caveolin-1 labeling at shallow caveolae (arrowheads) and in nonindented, intramembrane, particle-containing areas (arrows). All figures: freeze-fractured smooth muscle cells washed with SDS, immunostained with anticaveolin-1, and reacted with a colloidal gold marker. Original bars: 0.5 µm.

After immunostaining with anticaveolin-1, caveolin-1 is found on the P-face of the plasma membrane (Fig. 1A ). The E-face is always completely unlabeled. Deep caveolae of the P-face are generally strongly marked on their rims (Fig. 1B ), whereas labeling of shallow caveolae and of intramembrane particle-containing areas is weaker (Fig. 1C ). Some caveolin-1 labeling of the P-face of the plasma membrane occurs in morphologically undifferentiated regions, and some areas of the membrane are quite devoid of label (Fig. 1A ).

2. Caveolin-1 is located only in the E-faces (exoplasmic leaflets) of intracellular membranes
Immunostaining shows that caveolin-1 is also present in the membranes of most intracellular compartments. Intracellular membranes marked with anticaveolin-1 include those of endoplasmic reticulum (ER) cisternae (Fig. 2 ), trans-Golgi vesicles, cis-Golgi cisternae, and the inner and outer nuclear membranes, but medial Golgi stacks are essentially label-free (all not shown). Surprisingly and in contrast to the plasma membrane, caveolin-1 labeling is exclusively on the E-faces of the intracellular membranes; caveolin-1 never occurs on the P-faces of the membranes of intracellular compartments.

View larger version (230K): [in this window] [in a new window]   Figure 2. Caveolin-1 is found only on the E-faces (exoplasmic leaflets) of ER membranes. Colloidal gold particles label caveolin-1 on exposed E-faces (E) but not P-faces (P) of ER cisternae membranes. Free caveolin-1 occurs in the lumens of some cisternae of the ER (arrows).

We also found free caveolin-1 in the lumens of ER cisternae (Fig. 2) and in lipid droplets (not shown).

CONCLUSIONS AND SIGNIFICANCE

Caveolin-1 labeling always occurs on the P-face, never on the E-face, of the plasma membrane but only on the E-faces, never on the P-faces, of membranes of intracellular compartments and organelles, including trans-Golgi vesicles, cis-Golgi cisternae, ER, and the inner and outer nuclear membranes. Thus, caveolin-1 resides in the cytoplasmic leaflet of the plasma membrane but in the exoplasmic leaflets of membranes of intracellular compartments. We conclude that caveolin-1 transits the membrane bilayer at some as-yet unidentified station on its itinerary through the cell. To find out where this occurs will surely be of great significance for understanding caveolin-1 cycling, cholesterol transport, and protein translocation across membranes.

The differential disposition of caveolin-1 in cell membranes has additional implications. Underlying the exchange of sides of the membrane may be some previously unrecognized phenomenon of real biologic interest. At the very least, the finding of sidedness of caveolin-1 distribution in membranes makes current hypotheses of caveolin-1 cycling, cholesterol transport, and lipid droplet formation virtually untenable. Why? Whenever membranes fuse, the cytoplasmic leaflets of the membrane partners invariably coalesce together, and exoplasmic leaflets coalesce with exoplasmic leaflets; nonadherence to this principle would inexorably traffic intramembrane proteins to the opposing membrane leaflet during fusion. Thus, the sidedness of caveolin-1 in plasma and intracellular membranes definitively precludes transport via the classic endocytotic pathway, as caveolin-1 en route to the plasma membrane would be delivered to the exoplasmic leaflet, where it is never found and vice versa (see Fig. 3 ). Clearly, a vesicle-free, cytosolic phase is always mandatory for caveolin-1 cycling and consequently, for caveolin-1-assisted cholesterol transport. In addition, lipid droplets, which are known to be enveloped with caveolin-1 molecules, cannot arise simply by bulging of the cytoplasmic leaflet of intracellular membranes as postulated by others, as caveolin-1 is located on the wrong (exoplasmic=luminal) side of the membrane bilayer. New explanations for these processes will have to be sought, and these should reconcile the enigmatic sidedness of caveolin-1 in cell membranes revealed here.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schemes of caveolin-1 cycling between the plasma membrane (PM) and membranes of intracellular compartments (IM). A) Currently hypothesized distribution of caveolin-1 in the cytoplasmic leaflets (red) of the PM and IM. B) True disposition of caveolin-1 in the cytoplasmic leaflet of the PM and in the exoplasmic leaflet (green) of IM as revealed by freeze-fracture immunolabeling. It is in this configuration that caveolin-1 cycles back and forth between IM and the PM, ostensibly crossing the lipid bilayer somewhere on its itinerary through the cell. C) As it is well established that cytoplasmic leaflets coalesce together, and exoplasmic leaflets coalesce together when membranes fuse, fusion of IM with the PM would deliver caveolin-1 to the extracellular leaflet of the PM. This does not happen, thus ruling out the classic endocytotic pathway for caveolin-1 cycling.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0008fje; doi: 10.1096/fj.03-0008fje

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