The organization of endoplasmic reticulum export complexes

After incubation in vitro in the presence of ATP and cytosol, semi-intact cells were fixed, rapidly frozen, and fractured to expose internal membranes. After etching and replication, intracellular organelles are rendered visible as three-dimensional structures. The surface of the ER was readily distinguishable from other subcellular compartments by the presence of ribosomes or, in the case of the nuclear envelope, additionally, nuclear pores. Adjacent to the surface of the ER, we frequently observed compact structures of similar size and apparent vesicular-tubular composition to VTCs observed in thin-sections ( Fig. 9 A) . These structures were completely absent in incubations that lacked cytosol or ATP. They varied in diameter, but were generally ~0.3-0.5 ~m across and ranged from a circular to a more oblong shape under normal incubation conditions. Assuming that the distinctive ~80-nm surface undulations correspond to vesicle profiles ( Fig. 9 A) and that a cluster can be represented as a sphere with a similar range of diameters, we estimate that VTCs detected in replicas could contain 50-110 individual elements. This value is compatible with the number of vesicular profiles determined by reconstruction from serial thin-sections.