Figure 6

The S. cereviciae Trs130 behaves as a membrane-associated, not trans-membrane, protein. A. Trs130 is found in the P100 fraction of cell lysates. Lysates (from 65 OD₆₀₀ cells) from wild type (NSY991) cells expressing Trs130-HA were fractionated by 100,000 × g centrifugation to yield supernatant, S100, and pellet, P100, fractions. The fractions (from 6.5 OD₆₀₀ cells) were tested by immuno-blot analysis using anti-HA antibody. B. Trs130 floats with membranes on OptiPrep gradients. The P100 fraction (from 50 OD₆₀₀ cells) from panel A, was loaded on an OptiPrep gradient. After centrifugation, the fractions were tested by immuno-blot analysis using anti-HA, anti-Ypt31 and anti-EMP47 antibodies. Trs130-HA fractionates with two other membrane proteins, Ypt31 and EMP47. C. Trs130 can be extracted from the P100 fraction by salt, but not by detergent. Resuspended P100 fractions (100 μg) were incubated with 0.5 M NaCl or 1% Triton X-100, and subjected to centrifugation. 50 μg were examined by immuno-blot analysis as described in panel A. Emp47 serves as a control for a trans-membrane protein that can be extracted by detergent, but not by salt. Bands were quantified and percent distribution in S100 and P100 is shown. Results in this figure are representative of three independent experiments.