Figure 5

Sro7p is found in both cytosolic- and membrane-bound pools. (A) Affinity-purified antibodies to Sro7p recognize a protein of ∼105 kD on SDS-PAGE. Cells from wild-type (SRO7, SRO77), single disruptants (sro77Δ or sro7Δ), double disruptants (sro77Δ, sro7Δ), and strains containing high copy SRO7 (2μ SRO7) were spheroplasted, lysed, and boiled in SDS sample buffer. The samples were examined by 7% SDS-PAGE gels followed by immunoblotting with affinity-purified α-Sro7p antibodies. The higher molecular mass forms present in the SRO7 high copy lane probably represent denatured aggregates since they are much less apparent when samples are diluted before boiling and do not change appreciably during pulse-chase experiments (data not shown). (B) Sro7p is found in the soluble and membrane fractions of the cell. Cells containing vector only (pB23) or SRO7 on high copy (pB497) were grown to logarithmic phase, spheroplasted, lysed, and spun to remove unbroken cells. The lysate was treated with detergent or a mock control and subjected to two successive centrifugations: 30,000 g for 15 min, and then the S30 supernatant was centrifuged at 100,000 g for 1 h. Pellet fractions were resuspended in the same volumes as the supernatants to normalize. Samples from each fraction were boiled, analyzed by 7% SDS-PAGE, and immunoblotted with affinity-purified α-Sro7p antibody. Samples were also run on a 12.5% gel and immunoblotted with α-Sso1/2p polyclonal antibody as an internal control of the fractionation procedure.