FIG. 6.
Depletion of Sec61p does not affect flippase activity. Wild-type and sec61ts cells were shifted to 38°C prior to preparation of microsomal membranes. (A) Sec61p is specifically depleted from P100 membranes. Membrane proteins of 38°C-shifted wild-type and sec61ts cells were analyzed by SDS-PAGE and immunoblotting using polyclonal antibodies against Sec61p. The blot was reprobed with polyclonal antibodies to the ER membrane protein Dpm1p in order to control for equal loading. (B) Stopped-flow kinetics of extraction of 1-palmitoyl-2-C6-NBD-PC from wild-type (squares) and Sec61p-depleted (circles) microsomal membranes (P100 membranes) by BSA. Labeled microsomal membranes were mixed with 1% (wt/vol) BSA (final concentration) using a stopped-flow accessory, and the fluorescence decay was recorded. Solid lines represent the fit of the experimental data to the three-compartment model diagramed in Fig. 2. The following values for the rate constants were obtained from this fit: for wild-type microsomal membranes, k+1 = 0.011 s−1, k−1 = 0.007 s−1, and k+2 = 0.029 s−1; for Sec61p-depleted microsomal membranes, k+1 = 0.014 s−1, k−1 = 0.009 s−1, and k+2 = 0.053 s−1. (Inset) Residuals for a fit of the data to the model and a monoexponential (Monoexp.) function of the extraction kinetics.