Biogenesis of transport carriers

Abstract

A chemical called Ilimaquinone (IQ) was identified based on its ability to convert the mammalian Golgi apparatus into small uniform size vesicles. We reasoned IQ hyper activates a membrane fission reaction and thus suitable for understanding this process. This reaction required a serine/threonine kinase called PKD. Surprisingly, PKD was found to control the biogenesis of transport carriers at the Trans Golgi Network (TGN) that are destined to cell surface. Other exit routes from the Golgi membranes were PKD independent. PKD binds diacylglycerol (DAG) and Arf1, and promotes the production of phosphatidylinositol‐4‐phosphate (PI4P). PI4P in turn recruits ceramide transfer protein (CERT) and the oxysterol binding protein (OSBP). The binding of Arfaptin1 to the TGN also requires PI4P. Arfaptin 1 contains a domain that has the ability to assemble into an amphipathic helix; PKD phosphorylates within this region and dissociates Arfaptin 1 from the TGN. PKD thus regulates membrane curvature by controlling the dynamics of the BAR domain containing Arfpatin 1 at the TGN. PKD also regulates the association of CERT and OSBP by phosphorylation. Altogether this suggests that PKD regulates the levels of PI4P, ceramide and sterols at the TGN, and important for transport carrier biogenesis. We have tested this hypothesis directly by affecting the levels of the ceramide product Sphingomyelin at the TGN. Perturbing the balance of Sphingomyelin inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. These findings highlight the role of lipid homeostasis in transport carrier formation. Altogether, the discovery of IQ as a Golgi vesiculating compound has been valuable for revealing the mechanism of transport carrier biogenesis during protein secretion.