FIGURE 4.

Nascent subunits lacking Rpl1 fail to recruit Mex67 efficiently. (A) Nmd3-TAP was purified from RPL1-repressed cells and from LMB-treated cells. Spectral counts/molecular weight (MW) for Arx1, Bud20, and Mex67 were normalized to Tif6 levels in each sample. TAP purifications were from AJY4008 treated with LMB for 30 min and from AJY4009 in which RPL1 was repressed for 1.5 h. (B) Western blots for Mex67, Rpl1, and Rpl8 in TAP purification samples from BY4741, AJY4008 treated with LMB for 30 min, AJY4009, AJY1874, AJY4001, AJY4012, and AJY4013 grown in galactose followed by 1.5 h glucose treatment (lanes 1–7, respectively). Mex67:Rpl8 and Rpl1:Rpl8 were calculated for each sample. Mex67:Rpl8 ratio in each sample was normalized to that in the LMB sample (lane 2), and Rpl1:Rpl8 ratio in each sample was normalized to that in the WT NMD3-TAP samples (lane 4). (C) Polysome profiles and western blots for monitoring sedimentation of Mex67 and Rpl8 done in the low salt buffer (50 mM K⁺) from extracts of WT (KBM13) and GAL::RPL1 (KBM20) cells, respectively, grown in galactose media followed by 2 h growth after adding glucose. (D) Mex67:Rpl8 in the fraction containing the 60S subunits was calculated for sucrose gradients of RPL1 expressed and repressed conditions in C. (E) Western blots for Mex67 and Rpl8 in extracts from KBM13 and KBM20 grown in galactose containing media for 48 h and then diluted and grown in fresh glucose-containing medium for 1.5 h.