The organization of endoplasmic reticulum export complexes

We have previously suggested that export from the E R is accompanied by concentration of VSV-G . This point was the subject of a recent debate (Balch and Farquhar, 1995; Griffiths et al., 1995a) . We have now applied an independent approach using quick-freeze, deepetch methodologies in conjunction with immunolabeling of VSV-G to generate three-dimensional replicas that allow us to directly assess the concentration of VSV-G in ER-derived vesicles. We found, on average, a value of ~800 gold particles per txm 2 in planar projection of clusters accumulated in either the absence or presence of GTP~S, or in the presence of the activated Sarl-GTP restricted mutant. The inclusion of the inhibitors prevents further rounds of vesicle budding, ensuring that we are examining concentration associated with export from the ER. A planar projection, however, is not a good approximation of the total surface area available on clusters for antibody binding. Since only the external surface of vesicles found on the perimeter of clusters is available for antibody binding Pind et al., 1994a ) (see Fig. 11 D) , a more reasonable estimate of VSV-G density can be determined if we assume that the antibody has access to the outer-half of a shell of 80-nm vesicles that occupy the perimeter of a 0.4-~m sphere (the size of a typical VTC). Compared with the surface area of the planar projection (~0.13 ~m2), the surface area of such a population of vesicles corresponds to a value of ~0.7 txm 2. This is an increase of approximately fivefold over the planar projection. Therefore the actual surface density of VSV-G in clusters observed in replicas is 800 gold particles per i~m 2 divided by 5 or 160 gold particles per ixm 2. This value, when compared to the average surface density of VSV-G before incubation in vitro (32 gold particles per ~m2), sug- Figure 11 . Diagram summarizing the three tiers of organization of ER export complexes in vivo (A) and in vitro (B). (A) An individual ER cisterna contains a collection of closely opposed buds that define a local transitional region (light zone with buds on the ER). This specialized region is dominated by the presence of COPII coats and is referred to as tier 1 (box outlined with dotted line). Tier II (cylindrical region outlined by dashed lines) includes buds on distantly connected ER strands that face a central VTC consisting of a collection of distinct vesicular-tubular elements that have COPI coats. Tier III includes the entire export complex and is outlined by the box with the solid line that encompasses both ER buds and a central VTC. The possible elevated concentration of COPII and COPI coat components within the local cytoplasm of export complexes is depicted by small dots and lines. (B) In semi-intact cells, there appears to be a more limited number of ER buds associated with the local transitional region defined by tier I (box outlined with dotted lines). The tier iI level of organization is completely missing in semi-intact cells, as the association of distantly connected ER strands appears to be lost during cell permeabilization. However, tier III (box outlined with solid line) is maintained, highlighting the juxtaposition of VTCs to buds on one ER strand. gests that VSV-G is concentrated five-to sixfold during budding from the ER. The fold-concentration detected here is very consistent with that observed previously using serial thin-sections Pind et al., 1994a) or in the present studies in the presence of the Sarl-G