MAL, an integral element of the apical sorting machinery, is an itinerant protein that cycles between the trans-Golgi network and the plasma membrane

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-D arby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane-mediated apical tra nsport. At steady state, MAL is predominantly located in perinuclear vesicl es that probably arise from the trans-Golgi network (TGN). To act on membra ne traffic and to prevent their accumulation in the target compartment, int egral membrane elements of the protein-sorting machinery should be itineran t proteins that cycle between the donor and target compartments. To establi sh whether MAL is an itinerant :protein, we engineered the last extracellul ar loop of MAL by insertion of sequences containing the FLAG epitope or wit h sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these m odified MAL proteins allowed us to investigate the surface expression of MA L and its movement through different compartments after internalization wit h the use of a combination of assays, including surface biotinylation, surf ace binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug tr eatments. Immunofluorescence and flow cytometric analyses indicated that, i n addition to its Golgi localization, MAL was also expressed on the cell su rface, from which it was rapidly internalized. This retrieval implies trans port through the endosomal pathway and requires endosomal acidification, be cause it can be inhibited by drugs such as chloroquine, monensin, and NH4Cl . Resialylation experiments of surface MAL treated with neuraminidase indic ated that similar to 30% of the internalized MAL molecules were delivered t o the TGN, probably to start a new cycle of cargo transport. Together, thes e observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling b etween the TGN and the plasma membrane.