nature06828 - Vol 452 | 10 April 2008 |...

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ARTICLES Clathrin is a key regulator of basolateral polarity Sylvie Deborde 1 , Emilie Perret 1 * , Diego Gravotta 1 * , Ami Deora 1 , Susana Salvarezza 1 , Ryan Schreiner 1 & Enrique Rodriguez-Boulan 1,2 Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells. Epithelial cells require a polarized distribution of their plasma mem- brane (PM) proteins to perform a variety of vectorial functions in absorption and secretion 1–3 . Generation of polarity requires mechan- isms to sort the PM proteins into apical and basolateral domains, separated by tight junctions. Sorting is directed by sorting signals in the PM protein. Basolateral sorting signals are distinct determinants in the cytoplasmic domain; in some cases they resemble tyrosine and dileucine motifs similar to those used at the cell surface for clathrin- mediated receptor endocytosis 4–7 . An epithelial-specific adaptor, AP1B 8,9 , localizes to recycling endosomes and mediates biosynthetic and/or post-endocytic sorting of basolateral PM proteins with tyrosine motifs (for example vesicular stomatitis virus (VSV) G protein (VSVG) and low-density lipoprotein receptor (LDLR)) and non-tyr- osine motifs (for example transferrin receptor (TfR)) 10–12 .AP1Bisa tetrameric adaptor with clathrin-interacting domains in its c subunit that co-localizes with clathrin-coated vesicles near the Golgi 13 . All of the above is compatible with a possible function of clathrin in the biosynthetic sorting and recycling of PM proteins. Indeed, an early cell-fractionation study detected a newly synthesized PM protein in clathrin-coated vesicles 14 . However, a requirement for clathrin in the biosynthetic route of PM proteins has never been shown. Acute cross- linking experiments failed to detect the involvement of clathrin in recycling of TfR to the PM of non-polarized cells 15 ,a l thoughmo re recent in vitro reconstitution experiments 16 and an RNA-mediated interference (RNAi) study 17 support the involvement of clathrin in non-polarized PM protein recycling. Here we study the involvement
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This note was uploaded on 11/01/2010 for the course PRC 1234 taught by Professor All during the Spring '10 term at HKU.

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nature06828 - Vol 452 | 10 April 2008 |...

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