Title: The organization of endoplasmic reticulum export complexes Document date: 1996_10_1
ID: xxlcdbqi_57
Snippet: To identify whether the above structures formed in the presence of cytosol and ATP contained ER-derived cargo proteins such as VSV-G, NRK cells were infected with ts045 VSV at the restrictive temperature. After permeabilization and incubation in vitro at 32°C, cells were immunolabeled for VSV-G using the immunodiffusion protocol and replicas were prepared. While a combination of prolonged incubation using mild fixation conditions to label VSV-G .....
Document: To identify whether the above structures formed in the presence of cytosol and ATP contained ER-derived cargo proteins such as VSV-G, NRK cells were infected with ts045 VSV at the restrictive temperature. After permeabilization and incubation in vitro at 32°C, cells were immunolabeled for VSV-G using the immunodiffusion protocol and replicas were prepared. While a combination of prolonged incubation using mild fixation conditions to label VSV-G and the presence of prominent "gold shadows" from the etching and replication reduces the ability of the technique to reveal surface features of these immunogold-labeled clusters, the distribution of VSV-G reveals the striking role of COPII in concentrative export. Before incubation of semi-intact cells at 32°C, gold detected on the surface of the ER (Fig. 10 A, arrowheads) and nuclear envelope (Fig. 10 B, arrowheads) was distributed in an apparent random manner throughout the ER cisternal network (Plutner et al., 1992; Balch et al., 1994) . The surface density of VSV-G in these ER membranes was 32 +-6 gold particles per p~m 2. In contrast, incubation for 45 min in the presence of cytosol and ATP led to a dramatic change in the distribution of VSV-G, rearranging gold particles to a limited number of VTCs that were well isolated from each other (Fig. 10 C) . The density of gold over VTCs projected as a flat surface parallel to the ER membrane (referred to as a planar projection) was ~850 -+ 250 gold particles per p~m 2, a value markedly higher than that observed in the plane of the ER membrane before incubation.
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