Figure 4.

Differential centrifugation of lysates derived from wild-type (NY10), sec6-4 (NY779), sec2-41 (NY132), and sec2-78 (NY1529). Cells were grown overnight in YP dextrose and then shifted to 37°C. After 2 h at the restrictive temperature, cells were harvested, and lysates (S1) were prepared as described in Materials and Methods. S1 supernatants were centrifuged at 10,000 g to generate supernatant S2 and pellet P2. The S2 supernatants were centrifuged at 100,000 g to obtain supernatant S3 and pellet P3. Equal volumes of samples were prepared for electrophoresis, separated on SDS-PAGE gels, and transferred to nitrocellulose. Western blots were probed with αSec4p polyclonal antibodies and iodinated protein A. No difference in the relative distribution of Sec4p between S3 and P3 in sec2-41 and sec2-78 compared to sec6-4 is seen. The shift of Sec4p from P2 to P3 in sec6-4 compared to wild-type is caused by vesicle accumulation.