Figure 4.

GFP-Aip3p is associated with microsomes. Wild-type cells (A–E) or the sec4-8 strain DBY5894 (F) transformed with the GFP-Aip3p–expressing plasmid pDAb204 (A, C, and D) or the GFP-expressing plasmid pTD125 (B) were grown at 30°C (A–D) or 37°C for 90 min (E and F) to 2 × 10⁷ cells/ml. Cells were collected, washed, and lysed in a French Press, and a sample was collected (T). The cell extracts were then centrifuged for 20 min at 15,000 × g, and a sample of the supernatant was collected (S1), the pellet was reconstituted in the original volume, and a sample was taken (P1). The S1 fraction was then centrifuged further at 100,000 × g for 1 h, and a sample of the supernatant was collected (S2). The pellet was reconstituted in the original volume, and a sample was taken (P2). The samples were separated on an SDS-PAGE gel, transferred to nitrocellulose, and blotted with anti-GFP antibodies (A, B, E, and F), anti-Pma1p antibodies (C), or anti-Snc1p antibodies (D).