Figure 6.

Gea1p and Gea2p are peripheral membrane proteins. (A) Strains that contained either a chromosomal C-terminal myc-tagged Gea1p or Gea2p were grown in YPD to early to mid log phase. Cells were harvested, gently lysed, and subjected to differential centrifugation. To enrich for Gea1p and Gea2p, we subjected the supernatant (S) and the solubilized pellets (P) to immunoprecipitation with anti-myc antibodies coupled to proteinA-Sepharose. The precipitates were analyzed by immunoblot decorated with anti-myc-antibodies. (B) Gea2p is peripherally associated with membranes. YAS61, which contains a chromosomal Gea2p-myc-his₆, was grown to early log phase. Cells were harvested and lysed with glass beads under native conditions. Aliquots of the lysate were supplemented with either 1 M NaCl or 0.2% Triton X-100 (TX-100) before differential centrifugation. Samples were analyzed by immunoblot with antibodies directed against the myc-epitope, Anp1p and Pgk1p.