Fig. 1.

Rps3 N-domain assembly promotes Hrr25 recruitment. a Reduced binding of Hrr25 to particles impaired in Rps3 N-domain/Rps20 contact formation. Tsr1-TAP particles isolated from wild-type cells or from cells expressing the indicated rps3 or rps20 mutant alleles were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. Mass spectrometric (MS) analyses revealed that, while Hrr25 is present on particles isolated from wild-type strains, it is absent or strongly reduced from particles on which the Rps3 N-domain is not assembled (i.e., rps3.K7/K10>ED and rps20.D113/E115>K). Asterisks indicate the band of the Tobacco etch virus (TEV) protease used to elute pre-40S particles from the IgG beads. b, c Characterization of the interaction between Ltv1 and Hrr25. A C-terminal Hrr25 truncation (Hrr25(1–394)) and a mutant version thereof (Hrr25(1–394).K38R) fused to the Gal4 DNA-binding domain (BD) was tested for Y2H interaction with Ltv1 (b) and Ltv1 phosphomutants (Ltv1.S6>A and Ltv1.S6>E) (c) fused to the Gal4 activation domain (AD). Cells were spotted in ten-fold serial dilutions on SDC-Leu-Trp, SDC-His-Leu-Trp (-his; growth on this medium indicates a weak interaction), and SDC-Ade-Leu-Trp (-ade; growth on this medium indicates a strong interaction) plates. d Ltv1 is required to recruit Hrr25. Tsr1-TAP particles were isolated from ltv1Δ cells expressing plasmid-borne wild-type LTV1, the ltv1(S336/S339/S342>A) phosphomutant, or harboring an empty plasmid (Δltv1). Eluates were analyzed by SDS-PAGE, Coomassie staining, and MS. The asterisk indicates the TEV protease. e Hrr25(1–394).K38R (left panel) and Enp1 (right panel) fused to the BD were tested for Y2H interaction with the indicated Ltv1 fragments fused to the AD. f The deduced, minimal binding sites for Hrr25 and Enp1 on Ltv1 are indicated. In addition, the sites for Rps3 binding, Rps20 binding, and phosphorylation by Hrr25¹⁹, as well as the nuclear export sequence³⁵, are depicted. g Enp1 overexpression weakens the Hrr25-Ltv1 interaction. Y2H strains expressing Hrr25(1–394).K38R fused to the BD in combination with the indicated Ltv1 constructs fused to the AD were transformed with empty plasmid (left panel) or with a URA3 plasmid for ENP1 overexpression under control of the ADH1 promoter (right panel). Cells were spotted in ten-fold serial dilutions on SDC-Leu-Trp-Ura (panel: -leu-trp), SDC-His-Leu-Trp-Ura (panel: -his), and SDC-Ade-Leu-Trp-Ura (panel: -ade) plates