ER/Golgi intermediates acquire Golgi enzymes by brefeldin A-sensitive retrograde transport in vitro

Secretory proteins exit the ER in transport vesicles that fuse to form vesi cular tubular clusters (VTCs) which move along microtubule tracks to the Go lgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell lin e, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was o ptimal when transport was blocked at an ER/Golgi intermediate step. The rap id drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for i n vitro complementation could be distinguished due to their different sensi tivities to brefeldin A (BFA). Transport occurred either by direct fusion o f preexisting transport intermediates with ER/Golgi intermediates, or it oc curred as a BFA-sensitive and most likely COP I-mediated step. Direct fusio n of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.