Figure 6

Functional comparison between yeast and human domain examples. Domain examples of Actin (A) and the histone core (B, scH2B) are chosen from the network peripheral domain category and PH domains (C) from the network core domain category. A. Yeast Actin (scActin) as an EGFP fusion protein was artificially expressed in the mouse NIH-3T3 fibroblast cells. Cells were stained with phalloidin (Texas-Red) and DAPI for polymeric F-Actin stress fibers and DNA (marks nucleus) respectively. Arrows in EGFP panel mark the "cable-like" distribution of scActin in the structures of polymeric Actin stress fibers illuminated by phalloidin. B. Similarly, the yeast histone protein (scH2B), also as an EGFP fusion, was expressed in NIH-3T3 cells. The expression of scH2B is completely restricted to the cell nucleus (marked by DAPI, and in composite panel). C. PH domains of human Akt1 (hsAkt1) and yeast Cla4 (scCla4) were purified as recombinant GST-fusion proteins. These proteins were used to probe the membrane-type PIP-strips in a protein-lipid overlay assay. Binding of PH domains to specific types of PtdIns-phosphates (left panels -- refer to the diagram and list for spot positions) was visualized through anti-GST-HRP far-western blots. Red arrows point to the binding of PH domains of hsAkt1 to PtdIns(3,4)P2 and ptdIns(3,4,5)P3 (highlighted in red circles in the diagram). Circled in light blue are the PtdIns-mono-phosphates that have no signaling roles in cells. Right panel: visualization of recombinant GST-PH domains of hsAkt1 and scCla4 (pointed by black arrows) on a coomassie blue gel.