The organization of endoplasmic reticulum export complexes

1-3, arrowheads), were rarely observed in vitro. Since previous studies used mild fixation conditions in conjunction with an immunodiffusion procedure to label VSV-G in VTCs , we reasoned that ER buds may be labile structures. We therefore applied more stringent fixation and embedding conditions to preserve ultrastrucrural details (see Materials and Methods). Under these conditions, ER buds were observed that had a characteristic coat resembling those found in vivo and were only detected adjacent to VTCs (not shown), suggesting that even in semi-intact ceils, budding is restricted to specialized regions of the ER, To examine the formation of buds and their relationship to VTCs in vitro, we made use of the nonhydrolyzable analog of GTP, GTP~/S, which permanently activates Sarl and other GTPases, leading to stable coat assembly and accumulation of buds. Interestingly, we observed for the first time using strong fixation conditions that nascent budding profiles generated in the presence of GTP~/S had not only a cluster appearance (Pind et al., 1994a) , but also frequently had a distinctive "beaded necklace" appearance with each vesicle being a bead (Fig. 7, B and C) . The vesicles were nearly identical in size but lacked luminal continuity. These strings of vesicles were similar to the shorter necklaces sometimes observed in vivo under normal incubation conditions (Fig. 7 A) . These necklaces were confined to only a small fraction of the total ER surface. By analyzing sections through >600 individual necklaces, we have never detected them to have more than one connection to the ER membrane, supporting the possibility that each string grows from a local area of budding activity. These observations suggest that budding continues Outer diameter of shell (lID) Figure 6 . Probability of a given bud having proximity to a second bud in the cell. Randomly chosen ER buds present in RBL cells were assigned as the center of reference, and distances between it and any other buds present in 30 consecutive serial sections were determined by building a series of concentric shells with a constant volume of 0.0042 ixm 3, corresponding to the volume of the first internal shell having a diameter 0.2 Ixm (to encompass a single bud) as described in the Materials and Methods. The probability was determined by counting the number of buds detected in each shell relative to the total number of buds detected (percentage of total). This value is plotted as shell number in which a second bud was found (open circles) or relative to the outermost diameter of a particular shell (closed circles). The calculated probability of a second bud having a completely random distribution in the cell (0.18 %) is presented for comparison (diamonds). in the absence of GTP hydrolysis, but that the vesicles fail to complete separation from one another. Upon immunolabeling, we found accumulated vesicles to be substantially enriched in VSV-G (Pind et al., 1994a) and components of both COPI (13-COP) (Pind et al., 1994a; Griffiths et al., 1995b) and COPII coats (Sec13 and Sec 23) (Fig. 8, A-C) .