Fig 9. Cdc28 binding to Yih1 fragments.

(A) Schematics of the GST-tagged Yih1 fragments used in this study. The N-terminal RWD domain and the C-terminal ancient domain are indicated, as well as the Yih1 region sufficient for actin, Gcn1, and ribosome binding (modified from [3]). On the left, the Yih1 region encompassing the major Cdc28 binding determinant, as found in this study is shown. The schematic is not to scale. (B) Identical amounts of total protein from WCEs of the gcn1Δ strain H2556 overexpressing the indicated GST-tagged Yih1 fragments, were subjected to GST-mediated pull-down assays as described earlier [3]. The precipitated material was subjected to SDS-PAGE and immunoblot using antibodies against Cdc28 and GST. The assay was conducted at least six times, and a representative result is shown. * indicates the location of the respective GST-fusion protein in the blot. (C) The amount of Cdc28 sequestered in B was determined by quantifying the signal intensity of the Cdc28 signals from at least six independent results, using the NIH Image J software. The values are shown relative to those found for whole cell extracts containing GST alone. (D) The overexpression levels of the Yih1 fragments are shown relative to the expression level of full length GST-Yih1. Modified from [3] (E) The relative binding strength of GST-fusion proteins to Cdc28 was calculated by dividing the relative amount of Cdc28 sequestration in C by the relative expression levels of the respective GST-fusion proteins in D. The values are shown relative to that of GST-Yih1.