Vectorial apical delivery and slow endocytosis of a glycolipid-anchored fusion protein in transfected MDCK cells.

Abstract

To characterize the mechanis that determine the apical polarity of proteins anchored by glycosylphosphatidyliuiositol (GPI), we studied the targeting of a GPIanchored form of a herpes simplex glycoprotein, gD-1, in transfected MDCK cells. Using a biotin-based targeting assay, we found that GPI-anchored gD-1 was sorted in llulay and delivered directly to the apical surface. Endocytodus of GPIanchored gD-1 occurred slowly and preferentially from the apical domain, while transcytosis of the basolateral fraction did not occur at a signifcant rate (incompatible with being a precursor to the apical poo). Prevention of tight junction formation by in on in medium with micromolar Ca2+ resulted in expression ofGPI-anchored gD-1 on the fee surface, but not on the attached surface of the cell. Our results indicate that the apical polarity of a GPI-anchored protein is generated by vectorial delivery to the apical membrane, where its distribution is maintained by slow endocytosis and by a retention system not necessarily involving the tight junction.

Intracellular Retention of Glycosylphosphatidyl Inositol-Linked Proteins in Caveolin-Deficient Cells

“…Then, the cells were scraped into 1 ml of ice-cold lysis buffer containing 10 mM Tris, pH 8.0, 0.15 M NaCl, 1% Triton X-114, and protease inhibitors and homogenized with a Dounce (five strokes). After 30 min on ice, the samples were briefly warmed to 37°C (for 2 to 3 min); this step allows the solubilization of GPI-linked proteins and causes the detergent Triton X-114 to undergo phase separation (45,51). The detergent phase was collected by centrifugation in the Microfuge (14, 000 ϫ g for 30 s) at room temperature; hydrophobic integral membrane proteins (including GPI-linked proteins) are known to partition almost exclusively into the lower Triton X-114 detergent phase (46,49).…”

“…However, it remains unknown how GPI-anchored proteins are then transported from lipid rafts at the trans-Golgi to lipid rafts at the cell surface. It has been proposed that caveola-like vesicles located at the level of the Golgi might act as exocytic vesicular carriers to transport GPI-linked proteins to the cell surface (45,54,78). However, evidence to directly support this hypothesis is lacking.…”

Apical trafficking in epithelial cells: signals, clusters and motors

“…Additional support for the raft hypothesis was provided by the finding that the transmembrane domain of the envelope glycoprotein HA of the apically budding influenza virus (Rodriguez-Boulan and Pendergast, 1980) seemed to specifically promote apical targeting through its affinity for DRMs (Lin et al, 1998;Scheiffele, 1997). Unlike in MDCK cells, in which GPI-APs are delivered vectorially to the apical surface (Lisanti et al, 1990;Paladino et al, 2006), in hepatocytes most GPI-APs are targeted first to the basolateral surface and subsequently transcytosed to the apical domain (Schell et al, 1992;Slimane et al, 2003). The underlying mechanisms are starting to emerge and are discussed later in this Commentary.…”

Glycosylphosphatidylinositol (GPI)-Anchored Proteins.

“…73) In the polarized cells such as MDCK cells, GPI anchor is supposed to function as a sorting signal for protein transport to the apical cell-surface. 74) In metazoa, however, a number of functionally diversified proteins such as enzymes, receptors and cell-surface antigens are anchored to the plasma membrane by GPI. Therefore, the exact roles of GPI anchor and GPI-anchored proteins remain unclarified.…”