Figure 4

Gag p6-S40 mutations increase binding to Alix in the yeast 2-hybrid assay. Panel A, Qualitative colony assay. Co-transformed plasmids encoding protein pairs were tested for interaction under permissive and selective conditions using double drop-out media (DDO, Trp and Leu) and triple drop-out media (TDO, Trp, Leu, His), respectively. All transformed cells grew on the DDO media, indicating that plasmid pairs were expressed; only cells expressing interacting pairs grew on the TDO media. Panel B, Quantitative liquid assay. Beta-galactosidase assays were used to measure the relative strength of the S40 mutants plus Alix interaction. Beta-galactosidase units were normalized to P7L paired with Alix. Tsg101 plus P7L and Alix plus P7L-Y36S are negative controls. The S40A and S40F results represent five independent clones tested in 5 independent assays. Panel C, The beta-galactosidase signal is undetectable when the S40 mutants are paired with Alix F676D. WT Gag, P7L-S40A and P7L-S40F were each paired with wild type Alix or its F676D mutant which does not bind to Gag. Alix paired with Y36S serves as the negative control. Two independent clones were run in duplicate for each pairing. All values were normalized to WT paired with Alix.