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. 2000 Nov;11(11):3859–3871. doi: 10.1091/mbc.11.11.3859

Figure 3.

Figure 3

The N-terminal domain of Sec62p harbors the major interaction site. (A) Cells expressing Nug-Sec62p and Sec63CRUp were transformed with one of the PGAL1-controlled constructs shown in Figure 2, and 105, 104, 103, and 102 cells were spotted onto medium containing galactose but lacking uracil, histidine, leucine, and tryptophan to select for the presence of the plasmids. Growth was recorded after 4 d at 30°C. (B) Cells that were assayed for binding were extracted with buffer containing 1% Triton X-100, and the proteins were separated by 12.5% SDS-PAGE and incubated with anti-ha antibody after transfer onto nitrocellulose. The closely spaced doublets of bands seen for Sec62p and its C-terminal truncations arise very probably by an alternative initiation from a second methionine in position 10 of Sec62p. (C) Cells expressing Nug-Sec62p and Sec63CRUp were transformed with SEC62-Dha under the control of the PMET25-promoter, and 104 cells were plated on medium containing 0, 35, or 70 μM of methionine and lacking uracil, tryptophan, histidine, and leucine to select for the presence of the plasmids. Colonies were counted after 7 d of growth at 30°C. The number of colonies (the average of three experiments) was arbitrarily set to 100 for the cells grown without methionine, and the numbers of the cells grown in the presence of methionine were adjusted accordingly. The amount of protein was estimated after cell extraction and immunblotting by quantitative chemiluminescence (the average of three experiments is shown in arbitrary units). (D) Same as in C but cells were expressing Sec62-Dha or one of the deletion constructs under the control of the PGAL1-promoter, and the cells were plated on medium containing galactose. The number of colonies (the average of five experiments) was divided by the cellular amount of Dha-fusion protein as estimated by two independent immunoblotting experiments and quantitative chemiluminescence with the help of the lumi imaging system. The obtained ratio was arbitrarily set to 100 for Sec62-Dha, and the ratios of all the other tranformants were adjusted accordingly.