Genetic Analysis of Rap1p/Sir3p Interactions in Telomeric and Hml Silencing in Saccharomyces Cerevisiae

We have identified three SIR3 suppressors of the telomeric silencing defects conferred by missense mutations within the Rap1p C-terminal tail domain (aa 800-827). Each SIR3 suppressor was also capable of suppressing a rap1 allele (rap1-21), which deletes the 28 aa C-terminal tail domain, but none of the suppressors restored telomeric silencing to a 165 amino acid truncation allele. These data suggest a Rap1p site for Sir3p association between the two truncation points (aa 664-799). In SIR3 suppressor strains lacking the Rap1p C-terminal tail domain, the presence of a second intragenic mutation within the rap1s domain (aa 727-747), enhanced silencing 30-300-fold. These data suggest a competition between Sir3p and factors that interfere with silencing for association in the rap1(s) domain. rap1-21 strains containing both wild-type Sir3p and either of the Sir3 suppressor proteins displayed a 400-4000-fold increase in telomeric silencing over rap1-21 strains carrying either Sir3p suppressor in the absence of wild-type Sir3p. We propose that this telomere-specific synergism is mediated in part through stabilization of Rap1p/Sir3p telomeric complexes by Sir3p-Sir3p interactions.     

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.     

Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae.

Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.