Mapping the ubiquitin-binding region in Pha13 by a yeast two-hybrid assay.

(A) Left panel indicating Pha13 putative functional domains (A20 and AN1) and the sites of truncation. The ubiquitin-binding region is mapped to the A20 domain of Pha13. Various combinations of Pha13 truncations cloned in pGBK vector were co-transformed with pGADPhaUb into yeast AH109 strain. The transformants were spotted on control plates (2DO: Leucine-Trptophan dropped out medium) and selective plates (3DO/X-Gal: Leucine-Trptophan-Histidine dropped out and X-Gal added medium), which were incubated at 30°C for 4 days before photography. Red stars indicate cysteine in A20 domain. Yellow stars indicate cysteine replaced with lysine. (B) In vitro pull-down assay on AtSAP5 (used as positive control) [44], Pha13 and Pha13 A20 mutant (Pha13A20m), with polyubiquitin chains.