Despite decades of research, apical sorting of epithelial membrane proteins remains incompletely understood. We noted that apical cytoplasmic domains are smaller than those of basolateral proteins; however, the reason for this discrepancy is unknown. Here we used a synthetic biology approach to investigate whether a size barrier at the Golgi apparatus might hinder apical sorting of proteins with large cytoplasmic tails. We focused on Crb3, Ace2 and Muc1 as representative apical proteins with short cytoplasmic tails. By incorporating a streptavidin-binding peptide, these proteins can be trapped in the endoplasmic reticulum until addition of biotin, which triggers synchronous release to the Golgi and subsequent transport to the apical cortex. Strikingly, increasing the size of their cytoplasmic domains caused partial mislocalization to the basolateral cortex and significantly delayed Golgi departure. Moreover, N-glycosylation of ‘large’ Crb3 was delayed, and ‘small’ Crb3 segregated into spatially distinct Golgi regions. Biologically, Crb3 forms a complex through its cytoplasmic tail with the Pals1 protein, which could also delay departure, but although associated at the endoplasmic reticulum and Golgi, Pals1 disassociated before Crb3 departure. Notably, a non-dissociable mutant Pals1 hampered the exit of Crb3. We conclude that, unexpectedly, a size filter at the Golgi facilitates apical sorting of proteins with small cytoplasmic domains and that timely release of Pals1, to reduce cytoplasmic domain size, is essential for normal Crb3 sorting.

尽管进行了数十年的研究，但上皮膜蛋白的顶端分选仍不完全清楚。我们注意到顶端细胞质结构域比基底外侧蛋白的细胞质结构域小;然而，这种差异的原因尚不清楚。在这里，我们使用合成生物学方法来研究高尔基体的尺寸障碍是否会阻碍具有大细胞质尾部的蛋白质的顶端分选。我们专注于 Crb3 、 Ace2 和 Muc1 作为具有短细胞质尾部的代表性顶端蛋白。通过掺入链霉亲和素结合肽，这些蛋白质可以被困在内质网中，直到添加生物素，从而触发同步释放到高尔基体并随后转运到顶端皮层。引人注目的是，增加它们的细胞质结构域的大小导致基底外侧皮层的部分错误定位，并显着延迟了高尔基体的离开。此外，“大”Crb3 的 N-糖基化延迟，“小”Crb3 分离成空间上不同的高尔基体区域。在生物学上，Crb3 通过其细胞质尾部与 Pals1 蛋白形成复合物，这也可以延迟离开，但尽管在内质网和高尔基体上相关，但 Pals1 在 Crb3 离开之前解离。值得注意的是，一个不可解离的突变体 Pals1 阻碍了 Crb3 的退出。我们得出结论，出乎意料的是，高尔基体的大小过滤器有助于对具有小细胞质结构域的蛋白质进行顶端分选，并且及时释放 Pals1 以减小细胞质结构域的大小，对于正常的 Crb3 分选至关重要。