LAUSR

Significance Epithelial cells perform critical protective, secretory, absorptive, and sensory functions, for which they require plasma membrane polarization into apical and basolateral domains. Impaired polarity causes cancer and developmental and degenerative disorders. Research on fundamental polarity mechanisms has been hindered by the paucity of model proteins and by the use of overexpression systems. Here, we introduce a high-throughput surface proteomics approach based on domain-selective biotinylation and quantitative mass spectrometry that provides candidate proteins to study polarity under normal expression levels. Using this approach, we described that clathrin adaptors mediate apical and basolateral distribution of surface proteins, expanding the traditional notion that clathrin adaptors mediate only basolateral polarity. Our results establish quantitative surface proteomics as a powerful tool to study epithelial polarity. The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.