Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2023.07.13.548868v1?rss=1

Authors: de Caestecker, C., Macara, I.

Abstract:
Apical sorting of epithelial membrane proteins is an essential process but remains incompletely understood. Apical cytoplasmic domains are significantly smaller than those of basolateral proteins; however, the reason for this attribute is unknown. We asked if a diffusion barrier at the trans-Golgi network might impede apical sorting of proteins with large cytoplasmic tails. We used Crumbs3 and Ace2 as example apical transmembrane proteins with short cytoplasmic tails. FKBP was attached to the C-termini for inducible dimerization to FRB-tagged proteins. A streptavidin-binding peptide on the extracellular domain traps the proteins in the endoplasmic reticulum (ER). Biotin addition triggers release to the Golgi, then departure in vesicles to the apical cortex. Increasing cytoplasmic bulk by dimerization to FRB-SNAPtag moieties significantly delayed departure. Crb3 binds through its cytoplasmic tail to the Pals1 protein, and although Crb3 and Pals1 are associated at the ER and Golgi, Pals1 disassociates before Crb3 departure. A non-dissociable mutant Pals1 impedes Crb3 exit. We conclude that small cytoplasmic domains facilitate apical sorting. Reducing cytoplasmic domain size by timely Pals1 release is essential for normal kinetics of Crb3 sorting.

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