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The hallmark event of the canonical transforming growth factor β (TGFβ) family signaling is the assembly of the Smad-complex, consisting of the common Smad, Smad4, and phosphorylated receptor-regulated Smads. How the Smad-complex is assembled and regulated is still unclear. Here, we report that active Arl15, an Arf-like small G protein, specifically binds to the MH2 domain of Smad4 and colocalizes with Smad4 at the endolysosome. The binding relieves the autoinhibition of Smad4, which is imposed by the intramolecular interaction between its MH1 and MH2 domains. Activated Smad4 subsequently interacts with phosphorylated receptor-regulated Smads, forming the Smad-complex. Our observations suggest that Smad4 functions as an effector and a GTPase activating protein (GAP) of Arl15. Assembly of the Smad-complex enhances the GAP activity of Smad4 toward Arl15, therefore dissociating Arl15 before the nuclear translocation of the Smad-complex. Our data further demonstrate that Arl15 positively regulates the TGFβ family signaling.

How Golgi glycosyltransferases and glycosidases (hereafter glycosyltransferases) localize to the Golgi is still unclear. Here, we first investigated the post-Golgi trafficking of glycosyltransferases. We found that glycosyltransferases can escape the Golgi to the plasma membrane, where they are subsequently endocytosed to the endolysosome. Post-Golgi glycosyltransferases are probably degraded by ectodomain shedding. We discovered that most glycosyltransferases are not retrieved from post-Golgi sites, indicating that retention rather than retrieval is the primary mechanism for their Golgi localization. We therefore used the Golgi residence time to study Golgi retention of glycosyltransferases quantitatively and systematically. Quantitative analysis of chimeras of ST6GAL1 and either transferrin receptor or tumor necrosis factor α revealed the contributions of three regions of ST6GAL1, namely the N-terminal cytosolic tail, the transmembrane domain and the ectodomain, to Golgi retention. We found that each of the three regions is sufficient for Golgi retention in an additive manner. N-terminal cytosolic tail length negatively affects the Golgi retention of ST6GAL1, similar to effects observed for the transmembrane domain. Therefore, the long N-terminal cytosolic tail and transmembrane domain could act as Golgi export signals for transmembrane secretory cargos. [ABSTRACT FROM AUTHOR]