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Description: FIGURE 3: Recombinant She2p binds to ER microsomes but not to mitochondria. A, pelleting of purified microsomes with recombinant She2p and GST. She2p or GST were mixed with ER membranes or buffer before centrifugation through a 1.2 m sucrose cushion. Equivalent amounts of the pellet (Pel), supernatant (Sup), or input material were analyzed by Western blotting against She2p, GST, or Sec61p. She2p is detectable in the pellet only in the presence of membranes. Spurious amounts of GST can be seen in the pellet fraction. B, the indicated amounts of She2p were incubated with 40 æ¸l of microsomes (corresponding to 40 æ¸g of membrane-associated protein). Input and bound She2p were analyzed by quantitative Western blotting, and the ratio of bound She2p signal versus input was quantified. The mean ratio (in percent) and S.D. of three experiments is displayed. C, purified microsomes or yeast mitochondria were mixed with She2p and pelleted through a sucrose cushion. She2p copellets only with microsomes. Sec61p served as an ER marker, whereas Mcr1p indicated mitochondria. In the absence of membranes, She2p can only be detected in the supernatant. Inp, input. Data are collected from Figure 3.
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