Fig. 8: The NTD-L is sufficient to confer the essential function of C11 in S. cerevisiae.

a Schematic of plasmid shuffling experiment. The haploid strain yGAKL, rpc11Δ::Kan^R complemented with a pRS316-S. pombe-C11 expression plasmid was transformed with pYX242 expression plasmids containing no insert (ev) or the six different constructs of S. cerevisiae (sc)C11 indicated above the panels in b. Transformants were grown, and counterselection was then applied against the spC11 URA3 plasmid in media containing 5-fluoroorotic acid (5FOA) expecting to obtain scC11 (WT) and any mutant construct (mut) that might confer viability. b Initial individual transformants obtained after steaking in patches to plates containing media lacking leucine and containing galactose (top row). Bottom row shows results of counterselection; colonies from the upper row were steaked to plates lacking leucine, containing galactose and 5FOA. c Time course of growth in liquid media of the isolated transformants indicated. Error bars reflect standard error SE (standard error = SD//√n); n = 4 for each time point representative of four biological replicates. d Growth toxicity assay for scC11 overexpression. The “wild type” laboratory strain BY4741 with an intact chromosomal RPC11 gene was transformed with GAL1 promoter-driven expression plasmid pYX113 with no inset (ev) or with inserts indicated to the left of the plates. Both plates contained synthetic complete (SC) media lacking uracil, the left plate contained glucose and the right plate contained 2% each of galactose and raffinose. e Western blot of whole-cell extracts from strains indicated above the lanes, developed with an antibody raised against S. cerevisiae full-length scC11. Lane 1: S. cerevisiae BY4741; lane 2: yGAKL (rpc11Δ, complemented by pRS316-GAL1-spC11; lane 3: yGAKL\SML1a (plasmid shuffle transformant with scC11); lane 4: yGAKL\SML2a: (plasmid shuffle transformant with scNTD-L); lane M: MW markers indicated in kD from an adjacent lane of the same gel. The open triangle to the left of lane 1 indicates a band corresponding to scC11. f Northern blot of total RNA, 20 μg (lanes 1, 2) and 10 μg (lanes 3, 4) from cells described in e, probed for scC11 mRNA (top panel), spC11 mRNA (2nd panel), and SRP ncRNA (3rd panel). The stable SRP ncRNA provides a size marker as 522 nt RNA, which was also transferred to the top panel. The open triangle to the left of the top panel points to endogenous scC11 mRNA whose estimated size with modest poly(A) would be ~590 nt. The asterisk at the second panel represents a size marker corresponding to ~510 nt. The bottom panel shows the high molecular weight region only of the ethidium bromide-stained gel prior to transfer, with 25S and 18S rRNAs indicated, to serve as quality control for the integrity of the RNA.