FIGURE 4.

Sucrose density gradient fractionation of Kre6-containing compartments. The cell lysate of the KRE6 wild-type was fractionated on an 18–60% (w/v) sucrose density gradient in the absence (A and C) or presence (B and D) of 10 mm EDTA. Cells were collected from an early-log phase random culture (A and B). The experiment was repeated using a synchronized culture in which more than half of the cells had buds smaller than 20% of the mother cell size (C and D). After centrifugation, 12 fractions were collected from the top of the gradient, and aliquots were analyzed by SDS-PAGE and immunoblotting using anti-Kre6, anti-Scs2, and anti-Gas1 antibodies. The signal intensity of indicated proteins was quantified with ImageJ software and graphed using Microsoft Excel. The top of the gradient corresponds to fraction 1.