Determining the Functions of the Cytosolic Domains of mu1B

Shelby King

doi:https://doi.org/10.21985/N2QM88

Work

Determining the Functions of the Cytosolic Domains of mu1B

Public Deposited

Download PDF

Download All Files (.zip)

Polarized epithelial cells are divided by tight junctions into an apical membrane domain, which faces the lumen of an organ, and a basolateral domain, which is in contact with underlying connective tissues. To establish and maintain polarity, newly synthesized and recycling transmembrane proteins are sorted to either the apical or basolateral domain based on sorting motifs encoded in their extracellular domains or cytosolic tails, respectively. Frequently, clathrin adaptor complexes recognize cytoplasmic tail signals. Epithelial cells express a tissue-specific, heterotetrameric clathrin adaptor complex, AP-1B, which mediates protein sorting to the basolateral membrane domain through cargo selection into nascent vesicles at an intermediate sorting compartment, the recycling endosome. AP-1B is remarkably similar to the ubiquitously expressed AP-1A complex, which functions at the trans-Golgi network to target proteins into the lysosomal pathway for degradation. The two adaptor complexes differ only in incorporation of the medium subunit, μ1A or μ1B, respectively. In determining how μ1B facilitates AP-1B localization to the recycling endosome for sorting of transmembrane proteins to the basolateral membrane, I have examined the C-terminus of μ1B, which upon recruitment of AP-1B onto membranes is exposed to the cytosol and is available for interaction with protein and lipid partners. In particular, the C-terminus of μ1B interacts with PI(3,4,5)P3, and this interaction is necessary for AP-1B recruitment to recycling endosomes. Cell lines that lack μ1B accumulate PI(3,4,5)P3 only at the plasma membrane, indicating a role for AP-1B in generating this lipid species at the recycling endosome. We show that AP-1B/μ1B interaction with PI(3,4,5)P3 is required for correct targeting of AP-1B-dependent cargo to the basolateral membrane. Additionally, the C-terminus of μ1B recruits subunits of the exocyst complex, enabling docking and fusion of AP-1B vesicles at the basolateral plasma membrane. Because the C-terminus of μ1B can interact with PI(3,4,5)P3 and recruit exocyst subunits from the cytoplasm, overexpression of the C-terminus of μ1B interferes with the functions of AP-1B in a dominant negative manner, causing missorting of AP-1B-dependent cargo. Moreover, these two roles for the C-terminus of μ1B appear to be the main reason for its dominant negative function, as expression of a construct containing mutations in the C-terminus of μ1B that disrupt PI(3,4,5)P3 interaction and exocyst recruitment does not interfere with the correct functioning of AP-1B. By examining the C-terminus of μ1B, we have learned how AP-1B is able to mediate its specific function in targeting proteins to the basolateral membrane of epithelial cells.

Last modified