Using Separation-of-Function Mutagenesis To Define the Full Spectrum of Activities Performed by the Est1 Telomerase Subunit in Vivo

A leading objective in biology is to identify the complete set of activities performed by each gene. Identification of a comprehensive set of separation...

. Mutations in EST1 that were examined for ODN and/or LOF phenotypes.     Figure S1. Mutations in EST1 that were examined for ODN and/or LOF phenotypes.
(A) Schematic representation of ODN and/or LOF phenotypes resulting from mutations in individual amino acids, as indicated on the Est1 protein sequence. Examples of ODN phenotypes are shown in Figure 1A -1C and Supplemental Figure S1C, S1D and S2, and examples of LOF phenotypes are shown in Figure 1D -1G and Supplemental Figure  S1D and S3. Mutations that failed to show a phenotype are indicated by a black arrow; mutations that conferred a phenotype are indicated by arrows in a range of sizes to indicate the severity of the phenotype. Brown arrows highlight those allele where the LOF phenotype was more severe than the ODN phenotype (see also Supplemental Figure S1B and S1C); blue arrows correspond to the five mutations that are impaired for binding to TLC1.
(B) Assessment of Est1 protein levels, for mutant Est1 proteins that exhibited LOF phenotypes that were more severe than the corresponding ODN phenotypes (ODN and LOF data for these alleles are shown in Supplemental Figure S1C, S1D and S3K). Mutant Est1 protein levels were reduced to ~25 to 30%, relative to wild type Est1, for the Est1-R291E and Est1-F408D proteins, and 2-fold for Est1-W442D; protein levels were assayed in strains in which each mutation was integrated into the genome, to avoid plasmid-dependent variations in Est1 protein levels, and the anti-myc signal for Est1myc 12 , was normalized to myc 12 -Est2. The Est1-42 mutant protein, with amino acid changes (D287A, E290A, R291A, R292A) that partially overlapped with Est1-R291E, also showed a 4-fold reduction in protein levels. Genetic analysis of the est1-42 mutation was previously used to argue that this mutation was specifically impaired for the so-called "second function" of Est1 (Evans and Lundblad 2002); however, the results shown here argue that this "second function" phenotype was instead most likely due to reduced protein stability, rather than loss of a specific biochemical activity. A similar caveat applies to the interpretation of the in vivo role of Arg291 from a different study (Hawkins and Friedman 2014).

Figure S1
(continued). Mutations in EST1 that were examined for ODN and/or LOF phenotypes.
(C) Examples of mutations that exhibit weak ODN phenotypes in the yku80-∆ synthetic lethal assay (relative to the magnitude of the corresponding LOF phenotype). A yku80-∆ / p CEN URA3 YKU80 strain was transformed with 2 µ plasmids containing the indicated est1¯ mutation, which was expressed by the constitutive ADH promoter; serial dilutions of two independent transformants for each mutation were examined for viability on media that selected for the presence or loss of the YKU80 plasmid. The temperaturedependence of this synthetic growth phenotype provides a sensitive assay for detecting a broad range of ODN phenotypes, as previously described (Lee et al. 2008) and as illustrated by the est1-D513R mutation; this mutation confers an LOF phenotype that was indistinguishable from that conferred by est1-D510R and est1-F511D ( Figure 1E and data not shown), but an ODN phenotype that was reproducibly less pronounced, relative to that of est1-D510R and est1-F511D.
(D) Telomere length analysis in ODN and LOF assays, for six mutations that exhibit an attenuated ODN phenotype when compared with the corresponding LOF phenotype; est1-K444E and est1-R488E, which exhibit comparably strong ODN and LOF phenotypes, were included for comparison. ODN or LOF effects on telomere length (the top three panels and the bottom three panels, respectively) were assayed as described in Figure 1.  (B) Examples of three Est1 amino acids (Arg447, Arg485 or Phe511) that exhibited a pronounced ODN phenotype when mutated to a charged residue, but a much less severe ODN phenotype when mutated to alanine, consistent with our prior observations (Lubin et al. 2013). Telomere length of est1-∆ strains transformed with single copy plasmids with the indicated est1¯ mutations, expressed by the EST1 promoter, determined after ~75 generations of growth following transformation of the est1-∆ strain. Mutations that are boxed (panels A -E) are defective for RNA binding, as shown in Fig. 3. Mutations indicated by a red asterisk (panels E, G, P and Q) conferred a wild type telomere length phenotype, in contrast to what was observed in previous reports (Zhang et al. 2010;Tong et al. 2011;Sealey et al. 2011). Mutations with a blue asterisk (panel P) correspond to a cluster of residues proposed to mediate nuclear localization (Hawkins and Friedman 2014); we propose instead that the mutant phenotype more likely due to protein destabilization, based on the very weak ODN phenotype of est1-K455E (see Supplemental Figure S1C), relative to the more pronounced LOF phenotype for this mutation.
Hawkins, C. and K. L. Friedman, 2014. Normal telomere length maintenance in Saccharomyces cerevisiae requires nuclear import of the ever shorter telomeres 1 (Est1) protein via the importin alpha pathway. Eukaryot. Cell 13: 1036-1050.  (B) A darker exposure of the image shown in Figure 3A, providing further support for the inability of the C-terminal domain of Est1 to form a complex with TLC1 and Est2.
(C) Over-expression of RNA-binding-defective est1¯ mutations in a wild type yeast strain did not confer an ODN phenotype on telomere length, assayed as described in Figure 1.
(D) The position of the Est1 RBD identified in this study (indicated by a blue box on the schematic figure of Est1) contradicts a previous report, which relied on a three-hybrid approach to identify a different region of the fission yeast Est1 protein that bound the telomerase RNA (Webb and Zakian 2012. Two of the fission yeast residues implicated in this previously proposed fission yeast RNA binding activity (R194 and K252, highlighted in yellow) are poorly conserved, as shown by an alignment of Est1 proteins from budding and fission yeasts from this region. In addition, mutations in the equivalent residues (K223 and D281) in S. cerevisiae resulted in a wild type telomere length (right hand panel), further suggesting that these fission yeast residues do not define a conserved function. A third fission yeast residue (L48) identified in this prior study was in a region of the fission yeast Est1 protein with very limited sequence conservation, which precluded identification of the comparable residue for in vivo analysis in S. cerevisiae. In contrast, key residues in the Est1 RBD identified in this study are highly conserved from budding yeast to fission yeast (Fig. 3D).
longer exposure Figure S6. Additional analysis of the fourth function of Est1 (A) Anti-FLAG IPs, with the (FLAG) 3 epitope on Est3, reveals an altered Est1:Est2 ratio, which we have previously argued is due to a telomerase disassembly complex (Tucey and Lundblad 2014); this ratio is not disrupted in IPs from the est1-R488E strain, demonstrating that the disassembly pathway is not altered by this mutation.
(B and C) Anti-FLAG IPs from strains with (FLAG) 3 epitopes on Est2, Yku80, Ebs1, Sir4 or Pif1; Est1 and Est2 were tagged with (myc) 12 in all five strains. The amount of anti-Est2 IP was 0.1X of the amount loaded in the other lanes. (B) Est1 association with Est2 was clearly detected in the anti-Est2 IP lane, whereas following anti-FLAG IP of Yku80, Ebs1, Sir4 or Pif1, no association of these four proteins with either Est1 or Est2 could be detected, even in a substantially darker exposure of the anti-myc western. The handdrawn marks on the anti-myc westerns (indicated by dots) correspond to pre-stained 100 and 150 kDa size markers (visualized on the membrane after transfer). (C) Anti-FLAG westerns show that the (FLAG) 3 -tagged versions of Yku80, Ebs1, Sir4 or Pif1 were readily detectable in the anti-FLAG IPs, relative to the 10-fold reduction Est2 signal.
(E) A re-examination of the previously proposed interaction between Mps3 and Est1 (Antoniacci et al. 2007;Schober et al. 2009); anti-FLAG IP were performed as described for part (C), above, with a strain containing a (FLAG) 3 epitope on Mps3 and (myc) 12 epitopes on Est1 and Est2. As a control, 0.02X and 0.005X amounts of an anti-FLAG IP from a strain with the (FLAG) 3 epitope was on Est2 were included.