Figure 4. Physical interactions between ESCRT proteins and Ark1p as revealed by two-hybrid analysis.

(a–e) Budding yeast strains containing LEXA–galactosidase transcriptional readout were transformed with the yeast two-hybrid bait (LexA DNA binding domain) and prey (GAL4 transcription activation domain) constructs indicated. Ark1p was fused to LexA and ESCRT proteins from each class were fused to GAL4. Mbx1p, a known Plo1p interacting protein,²⁸ was used a positive control for the assay. Transformed strains were grown for three days on selective medium with the X-gal overlay assay then performed. Experiments were performed three times with qualitatively similar results and illustrative examples of performed two-hybrid reactions are shown. (f) Summary of physical interactions between Ark1p and ESCRT proteins identified by yeast two-hybrid analysis. The table indicates the presence or absence of yeast showing a blue colour observed when each of the ESCRT proteins with various versions of Ark1p was assayed.