PLASMA-MEMBRANE TARGETING, VESICULAR BUDDING AND RELEASE OF GALECTIN-3 FROM THE CYTOPLASM OF MAMMALIAN-CELLS DURING SECRETION

Galectin 3, a 30 kDa galactoside-binding protein distributed widely in epithelial and immune cells, contains no signal sequence and is exter nalized by a mechanism independent of the endoplasmic reticulum (ER)-G olgi complex. We show here that hamster galectin 3 overexpressed in tr ansfected cos-7 cells is secreted at a very low rate. A chimaera of ga lectin 3 fused to the N-terminal acylation sequence of protein tyrosin e kinase p56(lck), Nt-p56(lck)-galectin 3, which is myristoylated and palmitoylated and rapidly transported to plasma membrane domains, is e fficiently released from transfected cells indicating that movement of cytoplasmic galectin 3 to plasma membrane domains is a rate limiting step in lectin secretion. N-terminal acylation is not sufficient for p rotein secretion since p56(lck) and the chimaera Nt-p56(lck)-CAT are n ot secreted from transfected cells. The amino-terminal half of galecti n 3 is sufficient to direct export of a chimaeric CAT protein indicati ng that part of the signal for plasma membrane translocation lies in t he N-terminal domains of the lectin. Immunofluorescence studies show t hat Ntp56(lck)-galectin 3 aggregates underneath the plasma membrane an d is released by membrane blebbing. Vesicles of low buoyant density is olated from conditioned medium are enriched in galectin 3. The lectin is initially protected from exogenous collagenase but is later release d in soluble protease-sensitive form from the lectin-loaded vesicles. Using murine macrophages, which secrete their endogenous galectin 3 at a moderate rate especially in the presence of Ca2+-ionophores, we wer e also able to trap a galectin 3-loaded vesicular fraction which was r eleased into the culture supernatant.