Microscopy analysis of reconstituted COPII coat polymerization and Sec16 dynamics.

The COPII coat and the small GTPase Sar1 mediate protein export from the endoplasmic reticulum (ER) via specialized domains known as the ER exit sites. The peripheral ER protein Sec16 has been proposed to organize ER exit sites. However, it remains unclear how these molecules drive COPII coat polymerization. Here, we characterized the spatiotemporal relationships between the Saccharomyces cerevisiae COPII components during their polymerization by performing fluorescence microscopy of an artificial planar membrane. We demonstrated that Sar1 dissociates from the membrane shortly after the COPII coat recruitment, and Sar1 is then no longer required for the COPII coat to bind to the membrane. Furthermore, we found that Sec16 is incorporated within the COPII-cargo clusters, and that this is dependent on the Sar1 GTPase cycle. These data show how Sar1 drives the polymerization of COPII coat and how Sec16 is spatially distributed during COPII coat polymerization.