Sorting and trafficking of newly-synthesized transmembrane proteins and mechanisms for primary cilium exclusion or enrichment of apical transmembrane proteins

The sorting of membrane proteins at the trans-Golgi network was investigated using a novel technique based on immunoisolation of membranes with magnetic beads. Soluble factors thought to be involved in protein sorting and vesicle formation were shown to be recruited to rat liver Golgi membranes immunoisolated with beads. This technique was adapted to work with membranes from tissue culture cells and then applied to the question of whether membrane proteins traverse endosomes on the way from Golgi to plasma membrane. Capture of a newly-synthesized transmembrane protein soon after departure from the Golgi also captured proteins endocytosed from the cell surface, providing evidence that cargo can pass through endosomes on its way to the plasma membrane.;The primary cilium is an organelle that extends from the apical pole of polarized epithelial cells. The ciliary membrane has been described as subdomain of the apical membrane, with unique properties. Podocalyxin is an apical membrane protein that is excluded from the membrane of the primary cilium. We found that exclusion of podocalyxin from a subdomain of the apical membrane occurs before the appearance of the cilium and defines an early step in ciliogenesis. The cytoplasmic tail of podocalyxin contains a PDZ-binding motif that connects to the actin cytoskeleton and is required for podocalyxin's exclusion from the ciliary membrane domain. Deletion of podocalyxin's PDZ-binding motif increases podocalyxin's mobility in the plasma membrane and allows it into the cilium. NHERF-family protein EBP50 binds to podocalyxin's tail and is required for podocalyxin's link to actin. Transfer of the cytoplasmic tail of podocalyxin to an apical and ciliary membrane protein, CEACAM1, was sufficient to exclude CEACAM1 from the cilium. EBP5O's ERM-binding domain and ezrin's actin-binding domain could also be added to CEACAM1 to cause ciliary membrane exclusion. Conversely, CEACAM1 could be enriched in the cilium by addition of a microtubule-binding domain. Thus, membrane protein interaction with the actin cytoskeleton can result in exclusion from the primary cilium, whereas interaction with the microtubule cytoskeleton can result in enrichment in the cilium.