Figure 4.

Subcellular fractionation and solubilization of Bud1 from the particulate fraction. (A) Subcellular fractionation and solubilization of wild-type Bud1. Cells of yeast strain expressing HA-epitope-tagged Bud1 were broken open by bead beating and centrifuged at 500 × g to produce whole cell extract (wce); the wce was subsequently centrifuged at 10,000 × g to produce pellect (P) and supernatant (S) fractions (lanes 1 and 2). Aliquots of the pellet fractions (P) were washed with the same lysis buffer (lanes 3 and 4) or treated with 500 mM NaCl (lanes 5 and 6), 2% Triton (lanes 7 and 8), and 1% SDS (lanes 9 and 10) before centrifuging at 10,000 × g again (34). Both pellet and supernatant fractions from equal amounts of cells (OD₆₀₀ unit) were loaded on 10% polyacrylamide gel and subjected to immunoblot analysis using monoclonal antibodies against HA epitope to detect HA-tagged Bud1. As a control for fractionation, identical samples were also subjected to immunoblot analysis using antibodies against the yeast endoplasmic reticulum membrane protein Sec61 (36). (B) Subcellular fractionation of Bud1^G12V and Bud1^K16N. Cells of yeast strain expressing HA-epitope-tagged Bud1^G12V or Bud1^K16N were treated in the same way as described in A.