ASSOCIATION OF ENGRAILED HOMEOPROTEINS WITH VESICLES PRESENTING CAVEOLAE-LIKE PROPERTIES

We report here that the homeoproteins Engrailed-l and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electr on microscopy, we observed that chick-Engrailed-2 expressed in COS-7 c ells associates with membrane fractions that are characterized as cave olae, This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathri n coat and the presence of caveolin and cholera toxin-binding sites, T hese data are fully confirmed by subcellular fractionation experiments , which demonstrate that transfected chick-Engrailed-2 is present in l ow density membrane fractions that are resistant to Triton X-100, enri ched in caveolin and solubilized by the addition of a cholesterol-bind ing detergent, a set of properties highly characteristic of caveolae, The association of Engrailed-2 with specific membrane fractions observ ed after transfection in COS-7 cells is also observed for endogenous E ngrailed-l and Engrailed-2 expressed at late embryonic stages in the c erebellum and posterior mesencephalon of the rodent, Indeed, the two p roteins are present in membrane fractions that bear all the characteri stics of microdomains or caveolae-like domains, i.e. Triton X-100 resi stance, saponin solubilization, low density on sucrose gradients, enri chment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprote in F3/F11 and of the acylated growth-associated protein GAP-43, Finall y we demonstrate that part of the membrane-associated Engrailed, eithe r expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have bee n permeabilized with detergent, This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are al so associated with caveolae-like vesicles that are primarily transport ed anterogradely into the axon, and that they can get access to a comp artment compatible with secretion.