Caveolae Scaffolded Membrane Domains Rich in Raft Lipids

Caveolae were first defined morphologically by Palade, who observed plasma membrane invaginations in endothelial cells under the electron microscope [82]. He later named them "plasmalemmal vesicles" [83], implying that they would shuttle molecules across the cell. The name "caveolae" (little caves) was however coined two years later by Yamada, who described invaginations on the surface of gallbladder epithelial cells [84]. Although he did not distinguish between coated and uncoated invaginations, the name "caveolae" was later specifically attributed to flask-shaped invaginations of 50 to 100 nm diameter that were devoid of the cla-thrin-coat, but instead displayed a characteristic striated coat [85]. While research on clathrin-coated pits and vesicles was rapidly progressing, caveolae long remained elusive.

This was changed when, almost 40 years after the morphological description, caveolin was identified as the major protein constituent of caveolae [86, 87]. Subsequently, two additional caveolin genes were cloned, so that the original caveolin was from then on referred to as caveolin-1. Caveolin-2 was co-purified with cav-eolin-1 from adipocytes [88], and its expression pattern overlaps with that of cav-eolin-1. The two proteins are most abundant in endothelial cells, fibroblasts and adipocytes, and they form stable hetero-oligomeric complexes in vivo [89]. Cav-eolin-3 shows a high degree of sequence similarity with caveolin-1, but its expression is restricted to muscle cells in which there is low caveolin-1 expression [90]. Both caveolin-1 and -2 have a smaller b-isoforms in addition to the full-length a-isoform. Caveolin-1 assumes an unusual topology in that it is an integral membrane protein [91] but does not span the bilayer. Instead the central hydrophobic domain is thought to form a hairpin structure which inserts into the cytoplasmic leaflet, leaving both the N- and C-terminus in the cytoplasm [87].

A characteristic feature of caveolins is their propensity to form high molecular-weight homo- and hetero-oligomers. Highly stable caveolin-1 oligomers of 14 to 16 monomers, dissociating only upon harsh detergent treatment at elevated temperatures, were found to be assembled relatively rapidly after synthesis of caveolin-1 in the endoplasmic reticulum and prior to Golgi exit [92]. The domain responsible for the oligomerization was mapped to the N-terminus [93]. The N-terminus has also been shown to target caveolin-1 to caveolar invaginations at the plasma membrane, since its absence results in Golgi retention [94, 95]. This ensures that only caveolin oligomers, not monomers, are transported to the plasma membrane. In addition to homo-oligomerization, caveolin-1 can form similarly stable hetero-oli-gomers with caveolin-2, which are localized mainly to plasma membrane caveolae [89]. In the absence of caveolin-1, caveolin-2 is not able to oligomerize and is retained in the Golgi in the form of monomers and dimers [96-98], again indicating that only the oligomeric form is transported to the plasma membrane.

The fact that caveolin-1 immunostaining decorated the striated coat around plasma membrane caveolae [86], together with the observation that it self-assembled into filaments in vitro [92] indicated that it indeed was an integral coat component. Since then, the function of caveolae became very closely linked to the function of caveolin, and it was shown that formation of the stable plasma membrane invaginations depended on caveolin expression. Cells not expressing caveolin-1 (e. g., lymphocytes) lacked cell-surface caveolae, and the expression of caveolin-1 in these cells was sufficient to induce their formation [99]. Quantification of the number of caveolin-1 molecules per caveolae by fluorescence intensity distribution measurements revealed that the uniform size of caveolae as seen by electron mi croscopy results from a quantal assembly mechanism in which 144 ± 39 caveolin-1 molecules are incorporated into a single caveola [100]; caveolin-2 was not assessed in this study. Caveolin-1 filaments had previously been proposed to assemble from heptamers, measuring 10 nm in diameter [101]. If this model were true, then 144 caveolin-1 molecules would form a filament of roughly 200 nm length, enough to surround an invagination of 50-100 nm diameter with a circumference of 150-300 nm once. The structure and composition of the caveolar coat are far from being understood (see also Chapter 2) but, most likely, caveolin-1 is not the only coat component. Other open questions are, where is the coat assembled and what is the assembly mechanism?

Caveolin-1 has been shown to bind cholesterol and the ganglioside GM1, both in vitro and in vivo [102, 103]. Cholesterol-binding occurs with high affinity, resisting even harsh detergent treatments [103]. The lipid composition of caveolae is thus similar to that of lipid rafts, and it can be extrapolated that the caveolar membrane should also display properties of a liquid-ordered phase. However, a detailed lipid composition of isolated caveolae is still lacking. The strong interaction with two bona fide lipid raft components predisposes caveolin-1 for the role as a raft-clustering agent. Similar to clustered rafts, caveolae have been proposed to function as signaling platforms [104] (see also Chapters 5, 6, and 11). The clear parallels in lipid composition and the partial co-purification of lipid raft and caveolar components in DRMs [105, 106], or in membranes of low buoyant density [107], has often led to an equation of the two membrane systems. However, we will continue to refer to caveolae as plasma membrane invaginations scaffolded by the caveolin-coat. The stable membrane curvature of caveolae could be a result of two contributions. Curvature could be induced by: (1) the high cholesterol concentration [108]; and (2) the insertion of caveolin-1 into the cytoplasmic leaflet of the bilayer, which would increase the surface area of the cytoplasmic leaflet relative to that of the exoplasmic leaflet and thus promote inward bending of the membrane. This stabilization of a curved membrane structure and the presence of caveolins would distinguish caveolae functionally from lipid rafts.

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