Spatial partitioning of secretory cargo from Golgi resident proteins in live cells

We observed partitioning of the fluorescent Golgi resident T2-CFP and fluorescent cargo proteins VSVG3-YFP or VSVG3-SP-YFP upon Golgi exit after a synchronous pulse of cargo was released from the ER. Golgi elements remained stable in overall size, shape and relative position as cargo emptied. Cargo segregated from resident rapidly by blebbing into micron-sized domains that contained little or no detectable resident protein and that appeared to be continuous with the parent Golgi element. Post-Golgi transport carriers (TCs) exited repeatedly from these domains. Alternatively, entire cargo domains exited Golgi elements, forming large TCs that fused directly with the plasma membrane. However, domain formation did not appear to be an absolute prerequisite for TC exit, since TCs also exited directly from Golgi elements in the absence of large domains. Quantitative cargo-specific photobleaching experiments revealed transfer of cargo between Golgi regions, but no discrete intra-Golgi TCs were observed.Our results establish domain formation via rapid lateral partitioning as a general cellular strategy for segregating different transmembrane proteins along the secretory pathway and provide a framework for consideration of molecular mechanisms of secretory transport.During secretory transport, organelle resident proteins must separate from itinerant secretory cargo. In the Golgi apparatus, resident glycosylation enzymes sequentially modify secretory proteins after delivery from their site of synthesis in the endoplasmic reticulum (ER). Different classes of cargo are then sorted in the Golgi and delivered to specific final destinations – an intracellular compartment or the cell exterior – leaving the resident proteins behind. The Golgi itself comprises a collection of stacked membrane cisternae with a distinct architecture [1]. Golgi resident glycosylation enzymes are type II (N-terminus in the cytosol) transmembrane proteins with the catalytic domain extending into the Golgi