Figure 3.

(A) Emp46p is an integral membrane protein. Spheroplasts were prepared from cells (emp46 Δ emp47 Δ/3HA-EMP46/2myc-EMP47) and treated with buffer 88 (Buffer), 1% Triton X-100 (TX100), 0.5 M NaCl (NaCl), or 0.1 M sodium carbonate (pH 11) in buffer 88. Samples were centrifuged at 100,000 × g, and totals before centrifugation (T), supernatant (S), and pellet (P) fractions were resolved by SDS-PAGE and immunoblotted for Sec23p, Sec61p, anti-HA, or anti-myc. (B) Sucrose gradient fractionation of Emp46p, Emp47p, Kex2p, Pho8p, and Sec61p. A whole-cell lysate from cells expressing both 2myc-Emp47p and 3HA-YLR080w was separated on a 20–60% sucrose density gradient, and fractions were collected, starting with fraction 1 at the top. Fractions were probed by immunoblotting. All data are from a single gradient, plotted in two parts for clarity. Relative levels of Emp46p, Kex2p, Sec61p, Pho8p, and Emp47p as determined by densitometry of the immunoblots are shown in C. Twenty percent of a total lysate is shown as T.