Polarized cells such as neurons and epithelial cells maintain separate plasma membrane domains, each with a distinct protein and lipid composition, through intracellular sorting mechanisms that recognize classes of proteins and deliver them into separate vesicles for transport to the correct surface domain (1, 2). Sorting to the correct membrane is essential for the proteins to exhibit their biological functions, whereas missorting often results in pathological conditions (3, 4). The recognition event responsible for sorting has been under intense investigation for two decades, and a number of peptide sequences capable of specifying transport to the basolateral surface of epithelial cells (5-11), or cell body of neurons (12-16), have been characterized. All of these signals are located in the cytoplasmic domains of transmembrane glycoproteins. In addition to basolateral signals, three types of signals for sorting proteins to the apical surface of epithelial cells, or axon of neurons, are known. Glycolipid anchors direct proteins to the apical surface of several types of epithelial cells (17, 18), apparently by associating in the trans-Golgi network (19, 20) with detergent-insoluble membrane domains enriched in glycosphingolipids and cholesterol (21). Oligosaccharides on some secreted proteins appear to specify apical transport (22), although this mechanism does not apply to all secreted proteins (23-26).For many transmembrane glycoproteins, deletion of cytoplasmic sequences containing a basolateral sorting signal results in efficient transport of the protein to the apical surface, rather than the random transport expected for the deletion of specific sorting information (10,(27)(28)(29)(30). For other proteins, deletion of cytoplasmic sequences caused randomized transport, proving that transport to the apical surface does not occur by default (9, 31). In the reverse approach, introducing basolateral sorting signals into the cytoplasmic domain of the influenza hemagglutinin (HA) 1 was shown to have a dominant effect over apical sorting information (8, 31, 32) that has been recently localized to the transmembrane domain (8). These observations implied that some proteins carry apical sorting information that is recessive to cytoplasmic basolateral sorting signals. Basolateral signals could dominate over apical signals simply by being recognized earlier in the biosynthetic pathway, or sorting could occur in a common compartment where basolateral signals might bind tighter to the sorting machinery than apical signals. To investigate these questions, we attached a series of basolateral sorting signals to the strictly polarized membrane protein of small intestinal epithelial cells, lactasephlorizin hydrolase (LPH, EC 3.2.1.23-3.2.1.62), and determined their effect on the sorting of LPH.LPH, an integral type I membrane glycoprotein, is 1927 amino acids long containing a membrane anchor of 19 contiguous hydrophobic amino acids and a cytoplasmic domain of 26 amino acids. It is synthesized as a precursor with apparent mo...
The roles of various domains of intestinal lactasephlorizin hydrolase (pro-LPH) on its folding, dimerization, and polarized sorting are investigated in deletion mutants of the ectodomain fused or not fused with the membrane-anchoring and cytoplasmic domains (MACT). Deletion of 236 amino acids immediately upstream of MACT has no effect on the folding, dimerization, transport competence, or polarized sorting of the mutant LPH1646MACT. By contrast, LPH1646, an anchorless counterpart of LPH1646MACT, is not transported beyond the ER and persists as a mannose-rich monomer during its entire life cycle.The further deletion of 87 amino acids generates a correctly folded but transport-incompetent monomeric LPH1559MACT mutant. The results strongly suggest that dimerization and transport of pro-LPH implicate a stretch of 87 amino acids in the ectodomain between LPH1646MACT and LPH1559MACT. In addition, dimerization of pro-LPH requires at least two further criteria: (i) a correctly folded ectodomain of pro-LPH and (ii) the presence of the transmembrane region. Neither of these requirements alone is sufficient for dimerization. Finally, the sorting of pro-LPH appears to be mediated by signals located between the cleavage site of pro-LPH and the LPH1646MACT mutant.
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