The FAM Deubiquitylating Enzyme Localizes to Multiple Points of Protein Trafficking in Epithelia, where It Associates with E-cadherin and ß-catenin

Abstract

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, {beta}-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with {beta}-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with {beta}-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with {beta}-catenin and E-cadherin from a higher molecular weight complex (~500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, {beta}-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (~2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased {beta}-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and {beta}-catenin during trafficking to the plasma membrane.