Because SUMO is known to modulate biological processes by providing a binding surface for proteins that have the ability to bind to it, we considered the possibility that our ChIP results could be explained by a role of SUMO in enhancing a direct interaction between Tpz1 and Stn1/Ten1 . To test this idea, we conducted a series of yeast two-hybrid assays, using a strain that allowed us to monitor the activation of a HIS3 and a (more stringent) ADE2 reporter. Under the less stringent conditions (HIS selection), we were able to detect a weak interaction between full-length Stn1 and Tpz1 proteins, but not between Ten1 and Tpz1 (Fig 4A, rows 12 and 13). Although co-expression of Ten1 alongside the GBD-Stn1 fusion protein increased the strength of the interaction, now visible on plates lacking adenine as previously reported  (row 14), the data indicated that Stn1 is capable of interacting directly with Tpz1. Whether Ten1 associates with Stn1 to form direct protein contacts with Tpz1, or it might simply bind Stn1 and thus contribute to its stability and/or folding, is unclear. Previous work identified the region of Tpz1 between amino acids 224 and 420 as being minimally required for the binding of Tpz1 to Stn1/Ten1 . Because this domain overlaps K242, we investigated whether SUMO and a subdomain of Tpz1 can together constitute a Tpz1-interacting domain of higher affinity for Stn1. We first established that a Tpz1 domain truncated at the SUMOylation site (243–420) was capable of interaction with Stn1 to a level comparable to full-length Tpz1, either in the absence or presence of Ten1 (compare rows 9 and 12; and 11 and 14 in Fig 4). We then assessed the ability of SUMO (Pmt3) to bind Stn1 and found that the proteins do interact (Fig 4, row 1). Ten1, which on its own did not bind SUMO significantly (Fig 4, row 2), was able to strongly stimulate the interaction between Stn1 and SUMO (Fig 4, row 3), as it did for Stn1 and Tpz1. Interestingly, when SUMO was fused to the N-terminus of the Tpz1 243–420 domain, thus mimicking the naturally occurring SUMOylated Tpz1 protein, the interaction with Stn1 was enhanced, with respect to both the Tpz1 243–420 and SUMO proteins alone (Fig 4, compare row 4 with 9 and 1, respectively). The same was observed in the presence of Ten1 (Fig 4, compare row 6 with 11 and 3): the SUMO-Tpz1(243–420) construct in the presence of Ten1 conferred the strongest observed growth in the Stn1 interaction assays. Taken together, these results indicate that a central domain of Tpz1 encompassing amino acids 243–420 is sufficient to promote an interaction with Stn1 and that this interaction is stabilised by Ten1. In addition, SUMO is capable of interacting with Stn1 independently, and linkage of SUMO at its naturally occurring site in Tpz1 strongly enhances the ability of Tpz1 to interact with Stn1. This interaction can explain the effect of tpz1-Snm on the telomere association of Stn1/Ten1 (Fig 3E and F), and its significance is consistent with the increased level of the modification observed in late S phase (Fig 1C) concomitant with the observed time of recruitment of Stn1/Ten1 to telomeres .
Both the ChIP and yeast two-hybrid analysis strongly indicate that the function of Stn1 at fission yeast telomeres is modulated by SUMOylation. Because both methods assess physical interactions, within the telomeric complex and between proteins, we sought independent genetic evidence for this idea. We performed random-mutagenesis on stn1+, and produced an allele, stn1-75, which is defective for stn1 function at 36°C as judged by colonies growing poorly and turning deep red on phloxine plates at this temperature, indicative of likely telomere loss (Fig 4B, fourth row in each of the three groups of plates). The pot1-1 allele, which is also non-functional at 36°C and leads to telomere loss and chromosome circularisation, was used as a control . While the tpz1-Snm allele on its own did not confer any growth defect to cells (Fig 4, third row), sporulation of a heterozygous tpz1-Snm stn1-75 diploid (Fig 4B, tetrads 1–3) revealed that, remarkably, tpz1-Snm affected the viability of stn1-75 cells at 25°C, contrary to wild-type tpz1+ and consistent with the idea that SUMOylation of Tpz1 facilitates the telomere recruitment—and hence the function—of Stn1. In Fig 4C, we summarise the molecular interactions promoted by SUMOylated Tpz1 at telomeres as suggested by our findings.