levels. To evaluate whether the growth rate of the two deletion strains has any correlation with the L35 protein levels, we examined the polysome proﬁles of the deletion strains and compared them with that from an isogenic wild-type counterpart. As shown in Figure 1B, both Drpl35A and Drpl35B strains showed a deﬁcit of free 60S versus free 40S r-subunits, a mild decrease in 80S ribo- somes and polysomes, and most importantly, an accumu- lation of half-mer polysomes. Clearly, the Drpl35B strain led to less severe alterations of the proﬁles than the Drpl35A one. This result suggests that the reduced growth rate of the Drpl35A strain is due to a deﬁcit of 60S r-subunits, which is much larger than the one observed for the Drpl35B strain. To conﬁrm this, we quantiﬁed total r-subunits by using run-oﬀ and low-Mg2+ sucrose gradients. Indeed, in the Drpl35A strain we observed a 25% reduction in the overall quantity of 60S r-subunits relative to the wild-type strain whereas only a 7% reduction was detected in the Drpl35B strain (data not shown). In agreement with this net deﬁcit in 60S r-subunits, western blot analysis indicated that the L35 levels, as well as those of L1 were decreased in the Drpl35A strain, whereas the levels of the small subunit r-protein S8 were not signiﬁcantly aﬀected. No evident changes were observed in the Drpl35B strain (Figure 1C). Nucleic Acids Research, 2010, Vol. 38, No. 15 5181 We conclude that the diﬀerent growth defect observed in the Drpl35A and Drpl35B strains is due to the accom- panying deﬁcit in 60S r-subunits in each strain and indi- cates that RPL35A plays a more important role in the production of L35 than does RPL35B. Few extra copies of RPL35B or RPL35A, which were provided from centromeric plasmids, fully restored a wild-type growth rate and polysome proﬁle to the Drpl35A or Drpl35B strains, respectively (Supplementary Figure S5). This suggests that there is no functional distinction between the L35A and L35B proteins at least under standard la- boratory conditions when cells are grown in synthetic medium. Depletion of L35 leads to a strong 60S ribosomal subunit shortage To study the consequences of the L35 depletion we generated a conditional GAL::RPL35 strain (see ‘Materials and Methods’ section). Growth of this strain was identical to that of the isogenic wild-type strain on YPGal plates, showing that the production of N-terminally HA-tagged L35A derived from the GAL-RPL35A construct ensures wild-type growth (Figure 2A). As expected, only very residual growth of this strain was observed on YPD plates (Figure 2A). After shifting a mid-logarithmic culture of GAL::RPL35 Figure 2. Depletion of L35 results in a deﬁcit in 60S r-subunits. (A) Growth comparison of the strains BY4741 (Wild-type) and RBY175 (GAL::RPL35). The cells were grown in YPGal and streaked on YPGal (Gal) and YPD (Glc) plates, which were incubated at 30 C for 3 days. (B) The above strains were grown in YPGal at 30 C and shifted to YPD. Cells were harvested at the indicated times after the shift and cell extracts were prepared. Equivalent amounts of cell extracts were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8. (C) 10 A260 of cell extract from the GAL::RPL35 strain grown in YPGal (Gal) or shifted for 6 h (6 h Glc) or 12 h (12 h Glc) to YPD were subjected to polysome analysis in 7–50% sucrose gradients. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.