FIGURE 3.

The CTLH domain of Gid1 is necessary for interaction with Gid7, whereas the LisH domain is required for binding of Gid2, Gid8, Gid5, and Gid4. A, co-immunoprecipitation (IP) studies between the Gid1 mutants and Gid7 are shown. Cells were grown overnight in YPE and were then harvested either directly or 30 min after the addition of glucose. After glass bead lysis, Gid1 was precipitated using α-Myc-antibody and protein A-Sepharose. Precipitates were analyzed for the presence of Gid1 and Gid7 via Western blotting with α-Myc and α-HA antibodies. B, shown are co-IPs between Gid2 and the Gid1 mutants. In this experiment Gid2 was precipitated because HA3-tagged Gid2 has the same molecular weight as the heavy chain (HC) of the antibody. C–E, co-IPs between the Gid1 mutants and Gid8, Gid5, or Gid4, respectively, are shown. Because Gid4 only appears after the addition of glucose, samples were only taken 20 min after the shift. F, shown is a preliminary model of the Gid complex.