Mutational analysis of caveolin‐induced vesicle formation

Abstract

Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50^100 nm. Although recombinant expression of caveolin-1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin-caveolin interactions regulate the size of caveolae, as caveolin-1 undergoes two stages of self-oligomerization. To test this hypothesis directly, we have created two caveolin-1 deletion mutants that lack regions of caveolin-1 that are involved in directing the self-assembly of caveolin-1 oligomers. More specifically, Cav-1 v v61^100 lacks a region of the N-terminal domain that directs the formation of high molecular mass caveolin-1 homo-oligomers, while Cav-1 v vC lacks a complete C-terminal domain that is required to allow caveolin homo-oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav-1 v v61^100 and Cav-1 v vC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500^1000 nm. In addition, caveolin-induced vesicle formation appears to be isoformspecific. Recombinant expression of caveolin-2 under the same conditions failed to drive the formation of vesicles, while caveolin-3 expression yielded caveolae-sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin-1 expression, but continue to express caveolin-2, no morphologically distinguishable caveolae are observed. In addition, as caveolin-2 alone exists mainly as a monomer or homo-dimer, while caveolins 1 and 3 exist as high molecular mass homo-oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae-sized vesicles. In direct support of this notion, regulated induction of caveolin-1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin-2 to caveolae membranes. The ability of caveolin-1 to recruit caveolin-2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co-expressed and interact with each other to form high molecular mass hetero-oligomers containing both caveolins 1 and 2.z 1998 Federation of European Biochemical Societies.

Transgenic overexpression of caveolin-3 in skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype

“…3). This is consistent with previous observations demonstrating that recombinant expression of caveolin-3 in cultured cells is sufficient to drive caveolae formation (41).…”

Caveolin-2 Localizes to the Golgi Complex but Redistributes to Plasma Membrane, Caveolae, and Rafts when Co-expressed with Caveolin-1

“…In contrast to caveolin-2, caveolin-1 had a predominant plasma membrane localization in both polarized FRT and MDCK cells. Importantly, a substantial fraction of caveolin-2 changed its distribution from the Golgi complex to the plasma membrane in the presence of caveolin-1, in agreement with previous data that demonstrate an interaction of the two caveolins (8,10,29). This differential localization of caveolins -1 and -2 was striking because it was recently shown that in a nonpolarized fibroblastic cell line (3T3-L1 cells), epitope-tagged caveolin-2 and endogenous caveolin-2 are strictly co-localized with endogenous caveolin-1, both intracellularly and at the level of the plasma membrane (9,10).…”

Caveolin-3 Null Mice Show a Loss of Caveolae, Changes in the Microdomain Distribution of the Dystrophin-Glycoprotein Complex, and T-tubule Abnormalities

“…Recombinant expression of caveolin-3 in cultured cells is sufficient to drive caveolae formation (39). As caveolin-3 is the only member of the caveolin gene family expressed in striated muscle cells, caveolin-3 is thought to be responsible for caveolae formation in skeletal muscle cells in vivo.…”

“…Next, we evaluated if dystrophin and dystrophin-associated glycoproteins were enriched in cholesterol-sphingolipid-rich raft domains in WT and caveolin-3 null mice. When caveolin-3 is expressed, it is targeted to these lipid rafts and induces the formation of morphological caveolae (39), as caveolins are cholesterol-binding proteins (5,42). However, cholesterol-sphingolipid rafts exist in the absence of caveolin protein expression (30,33,43).…”