Structure prediction-driven genetics in Saccharomyces cerevisiae identifies an interface between the t-RPA proteins Stn1 and Ten1

Abstract

In Saccharomyces cerevisiae, Cdc13, Stn1, and Ten1 are essential for both chromosome capping and telomere length homeostasis. These three proteins have been proposed to perform their roles at chromosome termini as a telomere-dedicated t-RPA complex, on the basis of several parallels with the conventional RPA complex. In this study, we have used several approaches to test whether a predicted alpha-helix in the N-terminal domain of the S. cerevisiae Stn1 protein is required for formation of the proposed t-RPA complex, in a manner analogous to the comparable helix in Rpa2. Analysis of a panel of Rpa2-OB(Stn1) chimeras indicates that whether a chimeric protein contains the Rpa2 or Stn1 version of this alpha-helix dictates its ability to function in place of Rpa2 or Stn1, respectively. In addition, mutations introduced into a hydrophobic surface of the predicted Stn1 alpha-helix eliminated association with Ten1. Strikingly, allele-specific suppression of a stn1 mutation in this helix (stn1-L164D) by a ten1 mutation (ten1-D138Y) resulted in a restored Stn1-Ten1 interaction, supporting the identification of a Stn1-Ten1 interface. We conclude that Stn1 interacts with Ten1 through an alpha-helix, in a manner analogous to the interaction between the comparable subunits of the RPA complex.

Figure 1.—

In vivo assessment of Rpa2–OB^Stn1 chimeric proteins. The predicted structure of the N-terminal domain of the three chimeras are shown as ribbon representations, with the position of the domain relative to each full-length Rpa2–OB^Stn1 protein; Rpa2 and Stn1 sequences are depicted in blue or red, respectively. Viability of a rpa2-Δ strain (YVL2924) or a stn1-Δ strain (YVL2394), following eviction of a RPA2 or STN1 plasmid by passage on 5-FOA-containing media, is shown on the left and right half of each plate, as indicated; the ability of each chimera to rescue the lethality of rpa2-Δ or stn1-Δ is compared to control plasmids expressing the intact RPA2 or STN1 genes.

Figure 2.—

Mutagenesis of the predicted Stn1 α-helix disrupts interaction with Ten1. (A) Alignment of the predicted Stn1 α-helix, for 10 Stn1 proteins; amino acids 163–181 for the S. cerevisiae protein are shown. (B) Predicted structure of the α-helix of the S. cerevisiae protein, with the hydrophobic face indicated in yellow; see Figure S1 for a comparison with the comparable surface of the RPA32 α-helix, as well as an alignment of this region of RPA32. (C) FLAG–Stn1 (pVL2848, or the indicated missense mutations introduced into pVL2848) and Ten1 (pVL3115) were translated in a coupled transcription/translation reaction with ³⁵S-methionine, aliquots were subjected to FLAG-immunoprecipitation, and immunoprecipitates were resolved on a 15% SDS–PAGE gel, as described previously (Gao et al. 2007). (D) Yeast two-hybrid analysis of the interaction between Stn1 (wild-type or mutant versions of pVL859) and either Ten1 (upper; pVL2678) or Cdc13 (lower; pVL3125); dilutions of the S. cerevisiae strain pJ69–4A, coexpressing Gal4AD–Stn1 and either Gal4DBD–Ten1 or Gal4DBD–Cdc13, were plated on appropriate selective plates to monitor viability (not shown) and the ability to activate the GAL2–ADE2 reporter gene. (E) Viability of stn1⁻ missense mutations, expressed by the STN1 native promoter and on a CEN plasmid (wild-type or mutant versions of pVL1492), which were transformed into a stn1-Δ/p STN1 URA3 shuffle strain (YVL2394); serial dilutions were plated on selective plates to monitor total viable cells (not shown) and on 5-FOA-containing plates, to monitor viability following loss of the covering STN1 plasmid.

Figure 3.—

Mutations in the proposed Ten1-interacting domain of Stn1 result in telomere elongation. Telomere length analysis of a panel of stn1⁻ missense mutations introduced into pVL1492, and transformed into the stn1-Δ/p STN1 URA3 shuffle strain (YVL2394); strains were propagated for ∼25 generations following evicting of the covering STN1 plasmid, prior to preparing genomic DNA for analysis of telomere length.

Figure 4.—

Ten1 overexpression suppresses stn1–L164D and stn1–I168D. (A) The stn1-Δ shuffle strain (YVL2394), with a control vector (pVL248) or a vector expressing Ten1 by the strong constitutive ADH promoter (pVL3541), as well as pVL1492 (STN1), pVL3573 (stn1–L164D), pVL3577 (stn1–L164D), or YCplac111 (control vector); cultures of single colony isolates of strains expressing the indicated mutations were plated on selective media to monitor total viable cells and on 5-FOA-containing media to monitor viability following loss of the covering STN1 plasmid. (B) Telomere length analysis of the selected strains from A, analyzed after ∼25 generations following loss of the covering STN1 plasmid.

Figure 5.—

ten1–D138Y and stn1–L164D exhibit reciprocal cosuppression. (A) Viability of stn1-Δ ten1-Δ strains, containing pVL1492 (STN1) or pVL3573 (stn1–L164D) and pVL3779 (TEN1) and pVL3858 (ten1–D138Y), following loss of the covering STN1 TEN1 plasmid. In this experiment, single colony isolates of each strain were identified following propagation on 5-FOA-containing media, grown in liquid culture, and serial dilutions plated on rich media. (B) Western analysis of mutant or wild-type Stn1–(myc)₇ and Ten1–(FLAG)₃ proteins, following anti-FLAG immunoprecipitation. The Ten1–D138Y–(FLAG)₃ protein consistently displayed lower protein levels in immunoprecipitates, suggesting that this protein might be unstable; a longer exposure of the panel on the right is shown in Figure S4. (C) Photo micrograph of a stn1–L164D strain, following dissection of YVL3421, photographed after 3 days growth with a Zeiss Axioskop 50 with a Nikon Digital Sight DS-5M camera. (D) Single colony streak-outs of STN1 TEN1, stn1–L164D ten1–D138Y and stn1–L164D ten1–D138Y/p CEN ten1–D138Y strains, generated by dissection of YVL3422 transformed with pVL3858 (CEN TRP1 ten1–D138Y), propagated on rich media at 30° for 3 days; Figure S5 shows a comparison of these strains at 2 vs. 3 days. (E) A summary of (i) the viability of strains with mutations integrated at their corresponding genomic loci and (ii) co-immunoprecipitation data for the indicated wild-type and mutant proteins.

Figure 6.—

The proposed interaction between Stn1 and Ten1. A schematic representation is shown for the S. cerevisiae and S. castellii wild-type proteins, as well as for the double mutant S. cerevisiae stn1–L164D ten1–D138Y proteins. The position of residue L164 of Stn1 is shown on the basis of structure predictions of this region of the S. cerevisiae Stn1 protein (see Figure S1 and Figure S6); in contrast, the structure of the proposed Ten1 α-helix, and the proposed location of D138 on this helix in particular, is hypothetical, due to our inability to generate a reliable structural prediction for the S. cerevisiae Ten1 protein.