Fig 2.

Yeast phenotypes of mutants with altered RNA-binding sites of eIF4G2. (A) The expression of eIF4G2 mutants used in this study (YAS1955 derivatives) (Fig. 1A; also see Table S3 in the supplemental material). Twenty and 40 μg total protein from whole-cell extracts of the indicated strains were used for immunoblotting with anti-HA (to detect HA-eIF4G2) and antitubulin antibodies (loading control). (B) Temperature- and 3AT-sensitive growth of tif4632-7R was assayed by spotting 5 μl of 0.15 A₆₀₀ U culture (columns 1) and 10-fold serial dilutions (columns 10⁻¹ and 10⁻²) onto SC-His plates without (−) and with (+) 10 mM 3AT and incubated at the indicated temperatures for 3 days. (C) eIF4G2 mutations impair mRNA recruitment to the 40S subunit in vitro. Cell-free translation mixture with ³²P-labeled poly(A)-tailed MFA2 mRNA was fractionated by sucrose gradient velocity sedimentation. ³²P counts in relevant fractions are shown with an arrow indicating the fraction containing free 40S subunit. (D) Effect of tif4632-7R on GCN4-lacZ expression from p180. The graph summarizes β-galactosidase activity of YAS1955 (WT) and KAY901 (tif4632-7R [7R]) transformants carrying p180 in the presence (+) or absence (−) of 10 mM 3AT. The P value for differences between the values in columns 1 and 3 is presented.