Figure 2.

SVs accumulated in 38°C-shifted sec6-4 cells contain NBD-phospholipid translocase activity. (A) Membranes enriched in SVs were prepared from 38°C-shifted sec6-4 cells by differential centrifugation, labeled with NBD-lipids, and incubated for 3 h at 25°C without ATP. The reaction was then split, ATP was added to one-half, and the incubation was continued an additional 2 h. At times indicated, the fraction of fluorescent lipid remaining in the cytosolic leaflet was determined by back-extraction using defatted BSA as described in Materials and Methods. Data shown are means ± SD of three independent experiments. (B) SV-enriched membranes prepared from 27°C-grown sec6-4 cells were analyzed for ATP-dependent NBD-lipid transport by BSA back-extraction as in A. Data shown are means ± SD of two independent experiments. Note that the membranes from 27°C-grown cells display an apparent reduction in the passive movement of all four NBD-lipids compared with membranes from 38°C-shifted cells. Given the assay conditions used, this may be explained by an overall increase in the rate of flip-flop because of a larger fraction of ER membranes in the 27°C membrane preparation (also see Figure 6A).