Figure 5.
Aip3p fractionates with a late secretory vesicle marker in a sorbitol velocity gradient. The sec6-4 pep4Δ strain HJY3 was grown to 1 × 107 cells/ml and shifted to 37°C for 2 h, and cells were collected. Cells were then spheroplasted and lysed in a Dounce homogenizer. The cell extract was clarified by centrifugation at 450 × g for 3 min, loaded onto the top of a 20–40% sorbitol gradient, and centrifuged at 71,000 × g for 1 h (A–D), or microsomes were isolated and loaded onto the top of a 20–40% sorbitol gradient and centrifuged at 71,000 × g for 1 h (E–G). Sixteen fractions were collected from the top of the gradient, and samples from these fractions were separated on SDS-PAGE gels, transferred to nitrocellulose, and blotted with anti-Aip3p (A and E), anti-Snc1p (B and F), and anti- Pma1p (C) antibodies. Densitometry was used to quantify the relative amounts of protein in each fraction (D and G).