The organization of endoplasmic reticulum export complexes

To correlate our morphological observations with previous in vitro biochemical studies, we used semi-intact NRK cells to examine the potential role of COPII components in the formation of ER-derived buds and VTCs (Plutner et al., 1992; Peter et al., 1993; Balch et al., 1994; Pind et al., 1994b) . Semi-intact cells incubated for 30 min at 32°C in Figure 4 . Three-dimensional reconstruction of a peripheral export complex. Membrane contours shown in Fig. 3 are illustrated at a magnification of 160,000. Transparencies containing the membrane contours were scanned, overlayed, and coaligned based on the position of mitochrondria and ER. Green denotes ER cisternae, blue denotes ER buds, and red denotes tubules and vesicles of VTCs. Vesicular membrane contours within the VTC whose luminal continuity to the surrounding ER membranes was evident in consecutive sections were denoted in green. More intense shades of the same color reflect distance from the uppermost section. The alveolate coats of ER buds, where evident, are dictated by stipples. Images are presented either individually with the section number indicated (bottom two rows), or as an overlay containing four images (left) for clarity or all eight images (right). The resulting reconstruction shows the clustered structure of a typical VTC, surrounded by ER-bearing buds occasionally penetrating the periphery of a VTC, as evidenced by the coated portions of ER tubules (blue) adjacent to VTC tubules (red). the absence of cytosol failed to generate detectable VTCs (not shown). This result is consistent with the fact that preexisting VTCs are unstable during permeabilization (Aridot et al., 1995) and that cytosol contains essential soluble components of the COPII machinery required for the export of cargo from the E R (Barlowe et al., 1994; Kuge et al., 1994) . VTCs generated in vitro in the presence of cytosol are nearly identical to those observed in vivo. We have previously shown that they are composed of a compact network of tubules and vesicles that lack direct luminal connections to E R membranes and frequently label positively for B-COP using immunoelectron microscopy Pind et al., 1994a) . However, ER-budding profiles, like those observed adjacent to VTCs in vivo (Figs.