The organization of endoplasmic reticulum export complexes

We have provided the first quantitative, stereological de-scription of the three-dimensional organization of cellular structures involved in transport of cargo from the E R to the Golgi apparatus. Export complexes have a hierarchial organization that can be conceptually divided into three tiers (Fig. 11 A) . The first tier (Fig. 11 A, dotted box) consists of closely adjacent buds on a single E R cisterna. Each can give rise to an individual string or group of ERderived buds containing COPII coats. These budding loci were limited to specific regions of the E R in vivo, suggesting the existence of a defined number of export sites in the living cell. The second tier (Fig. 11 A, cylindrical region outlined by dashed lines) comes from the observation that buds on one cisterna were often found in close proximity to budding profiles emanating from E R cisternae derived from distantly connected regions of the ER. The third tier of organization encompassing the entire export complex ( Fig. 11 A, solid box) includes ER-derived buds that face into a region housing a central VTC. Tubular elements within VTCs contain distinctive COPI coats and are luminally discontinuous with the ER. While professional secretory Figure 10 . VSV-G is concentrated in ER-derived vesicles and VTCs. NRK cells infected with ts045 VSV at the restrictive temperature (39.5°C) to retain VSV-G in the ER were permeabilized with digitonin (Plutner et al., 1992) and either fixed immediately (A and B) or incubated for 45 rain at the permissive temperature (32°C) in the presence of cytosol and ATP (C), or additionally supplemented with 1 IxM Sarl[H79G] (E). After incubation, cells were fixed and VSV-G labeled with 10 nm (A-C, and E) or 6 nm (D) gold particles using the immunodiffusion protocol as described in the Materials and Methods. Cells were either prepared for thin-section TEM (D) or for quickfreeze, deep-etch replication (A-C, and E). At the restrictive temperature, VSV-G was uniformly distributed within the ER membrane (A) or the nuclear envelope (B). After a shift to the permissive temperature (B-E), VSV-G was concentrated in newly formed 80-nm vesicles associated with export complexes. (Arrowheads) Location of 6 nm (D) or 10 nm (A-C, and E) gold particles corresponding to the distribution of VSV-G. Due to the use of an immunodiffusion protocol before preparation of replicas and the high density of label, extended shadows from the gold particles partially obscure membrane outlines. Bar, 0.1 I~m. cells such as those found in the pancreas confine export predominately to a single transitional region juxtaposed to the cis face of the Golgi apparatus (Palade, 1975) , the two different cell lines used in the present study were found to have export complexes distributed throughout the cytoplasm. Our studies provide evidence that budding from the E R occurs in areas of intense morphological specialization. Each level of organization is discussed in detail below.