Fig 5. Acl4 interacts with the C-terminal part of the long internal loop of Rpl4.

A, Mapping of the Acl4 binding site on Rpl4a by the yeast two-hybrid (Y2H) interaction assay (left panel). Plasmids expressing full-length Rpl4a or the indicated Rpl4a fragments, fused to the C-terminal Gal4 DNA-binding domain (G4BD), and full-length Acl4, fused to the C-terminal Gal4 activation domain (G4AD), were co-transformed into the Y2H reporter strain PJ69-4A. Cells were spotted in 10-fold serial dilution steps onto SC-Leu-Trp (-LT), SC-His-Leu-Trp (-HLT), and SC-Ade-Leu-Trp (-ALT) plates, which were incubated for 3 d at 30°C. The different Rpl4a constructs are schematically depicted on the right; the colour code to indicate the different features of Rpl4 is as in Fig 1C. Close-up view of the long internal loop of Rpl4 (right panel). The minimal Acl4 binding site on Rpl4a, as determined by Y2H and in vitro binding assays, is coloured in red (amino acids 88–114; Y2H) and orange/red (amino acids 72–114; in vitro), respectively. B, In vitro binding assay between Rpl4a and Acl4. The indicated C-terminally (His)₆-tagged Rpl4a variants and full-length Acl4-Flag were co-expressed in E. coli and purified via Ni-affinity purification. Proteins were revealed by SDS-PAGE and Coomassie staining (top) or by Western blot analysis using anti-Flag (Acl4-Flag) and anti-His (Rpl4a-(His)₆ variants) antibodies (bottom). T, total extract; P, pellet fraction (insoluble proteins); S, soluble extract; E, imidazole eluate; M, molecular weight standard. Blue arrowheads highlight the bands corresponding to the different Rpl4a-(His)₆ variants used as baits for the purifications. Black arrowheads indicate the position of Acl4-Flag.