Fig 3. The C-terminal extension contributes to the efficient incorporation of Rpl4 into 60S subunits.

A, Expression of C-terminally truncated Rpl4a variants confers a dominant slow-growth phenotype. Empty vector (YCplac111) and plasmid-borne wild-type RPL4A or the indicated rpl4a deletion mutants, expressed under the control of the cognate promoter, were transformed into the haploid wild-type strain YDK11-5A. Transformants were restreaked and cells were spotted in 10-fold serial dilution steps onto SC-Leu (-Leu) plates, which were incubated for 3 d at 30°C. B, Analysis of the incorporation of C-terminally truncated Rpl4 variants into 60S subunits and translating ribosomes. Whole cell lysates, derived from wild-type cells expressing N-terminally TAP tagged Rpl4a or the indicated Rpl4a deletion variants, were prepared under polysome-preserving conditions in the presence of cycloheximide and analyzed by sucrose gradient centrifugation and fractionation. Five A₂₆₀ units were resolved in 10–50% sucrose gradients and the absorption profiles were recorded by continuous monitoring at A₂₅₄ (upper panels). Sedimentation is from left to right. The peaks of free 40S and 60S subunits, 80S free couples/monosomes, and polysomes are indicated. Half-mers are highlighted by arrowheads. Gradient fractions were subjected to Western blot analysis using anti-protA and anti-Rpl3 antibodies (lower panels). The asterisk indicates the residual NTAP-Rpl4a.N264 signal in this anti-Rpl3 Western blot.