Fig. 1. Mutations of the Stn1 SIM domain lead to elongated telomeres.

(A and C) Y2H analysis of budding yeast strain carrying the indicated plasmids. The indicated proteins were fused either to B42 activating domain (B42-AD) or to LexA DNA binding domain. Cells were grown and spotted on selective medium (-TRP and -HIS) to select the two Y2H plasmids and tested for the activation of either Leu2 or LacZ reporters. (B) Schematic representation of the Stn1 and Ten1 proteins. The NStn1 contains the OB-fold domain that interacts with Ten1, whereas the CStn1 contains the WH1-2. The SIM domain that starts at position 226 is embedded into the WH1. The IRQM and the IIYL motifs mutated in the stn1-1 and stn1-195 alleles are also represented, respectively. (D) Analysis of viability of the indicated strains at different temperatures. Left: Cells were grown at 25°C, then serially diluted (fivefold), and plated on YES-rich medium either complemented or not with phloxine. Pink color reflects impaired growth. The stn1-226 allele carries mutations in SIM (²²⁶ILAL²²⁹ to ²²⁶AAAA²²⁹). Right: Growth curve of WT and stn1-226 strains. Both strains were cultured at 25°C in YES medium and then shifted to 36°C for 24 hours. (E) Genomic DNA from cells with the indicated genotype was prepared, digested with Apa I, and analyzed by Southern blotting with a radiolabeled telomeric DNA probe. (F) Stn1/Ten1 interaction is analyzed by coimmunoprecipitation (co-IP). Pulldown was carried out using anti-Flag (M2, mouse monoclonal antibody) from cell extracts prepared from indicated cultures carried out at 25°C if not specified. The co-IP of Stn1 was monitored using anti-myc (9E10, mouse monoclonal antibody). (G) Ten1-Stn1 interaction analyzed by Y2H. Cells were grown and spotted on selective medium (-TRP and -HIS) and tested for activation of LacZ reporter. Image is displayed on black and white background to visualize the blue color. (H) Ten1Stn1 interactions with SUMO, SUMO-Tpz1, and Tpz1 analyzed by Y2H. Cells were grown and spotted on selective medium (-TRP and -HIS) and tested for activation of Leu2 and/or LacZ reporters.