Biochemical Properties of MAL and MAL2 that Confer Specificity to Apical Delivery Pathways
My focus is to identify regulators of apical delivery in polarized epithelial cells. Each plasma membrane (PM) domain of a polarized cell performs specific functions and has a unique distribution of proteins and lipids. In simple epithelial cells, newly synthesized apical proteins take a direct route to the apical plasma membrane from the trans-Golgi network. In contrast, apical proteins in hepatocytes take an indirect transcytotic route via the basolateral membrane. Myelin and lymphocyte protein (MAL) and MAL2 have been proposed to function in direct and indirect apical targeting, respectively. Hepatocytes lack endogenous MAL consistent with the absence of direct apical targeting. Does MAL expression reroute hepatic apical residents into the direct pathway? We found that MAL expression in WIF-B cells induced the formation of cholesterol and glycosphingolipid-enriched Golgi domains that contained glycosylphosphotidyl- inositol (GPI)-anchored and single transmembrane domain (TMD) apical proteins; polymeric IgA receptor (pIgA-R), polytopic apical, and basolateral resident distributions were excluded. Basolateral delivery of newly synthesized apical residents was decreased in MAL-expressing cells concomitant with increased apical delivery; pIgA-R and basolateral resident delivery was unchanged. These data suggest that MAL rerouted selected hepatic apical proteins into the direct pathway. Recently, it was proposed that lipid-raft microdomains are too small and transient to host apically destined cargo, and that lipid-associated proteins might serve to stabilize raft-sorting platforms. Do MAL and MAL2 promote raft-stablilization and clustering? Examination of lipid-association properties revealed that MAL is raft-associated, while MAL2 is not. Does MAL and MAL2 overexpression promote lipid-association of apical proteins? MAL overexpression promoted lipid-association of both single TMD and GPI-anchored proteins whereas, MAL2 did not. Do MAL and MAL2 oligomerize to promote raft-coalescence? MAL is an oligomer and its overexpression altered the oligomeric states of single TMD and GPI-anchored apical proteins. MAL2 is monomeric and upon pIgA-R overexpression, shifted to high molecular weight fractions in velocity gradients indicating complex formation. Together, these results suggest that MAL oligomerization and lipid association may promote raft clustering and stabilization at the TGN, whereas the mechanism by which MAL2 regulates transcytosis and basolateral delivery of pIgA-R remains elusive.
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