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A key unresolved issue in molecular evolution is how paralogs diverge after gene duplication. For multifunctional genes, duplication is often followed by subfunctionalization. Subsequently, new or optimized molecular properties may evolve once the protein is no longer constrained to achieve multiple functions. A potential example of this process is the evolution of the yeast heterochromatin protein Sir3, which arose by duplication from the conserved DNA replication protein Orc1 We previously found that Sir3 subfunctionalized after duplication. In this study, we investigated whether Sir3 evolved new or optimized properties after subfunctionalization . This possibility is supported by our observation that nonduplicated Orc1/Sir3 proteins from three species were unable to complement a mutation in To identify regions of Sir3 that may have evolved new properties, we created chimeric proteins of ScSir3 and nonduplicated Orc1 from We identified the AAA+ base subdomain of KlOrc1 as insufficient for heterochromatin formation in In Orc1, this subdomain is intimately associated with other ORC subunits, enabling ATP hydrolysis. In Sir3, this subdomain binds Sir4 and perhaps nucleosomes. Our data are inconsistent with the insufficiency of KlOrc1 resulting from its ATPase activity or an inability to bind ScSir4 Thus, once Sir3 was no longer constrained to assemble into the ORC complex, its heterochromatin-forming potential evolved through changes in the AAA+ base subdomain.

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The polyamines putrescine, spermidine, and spermine are required for normal eukaryotic cellular functions. However, the minimum requirement for polyamines varies widely, ranging from very high concentrations (mM) in mammalian cells to extremely low in the yeast Saccharomyces cerevisiae. Yeast strains deficient in polyamine biosynthesis (spe1⌬, lacking ornithine decarboxylase, and spe2⌬, lacking SAM decarboxylase) require externally supplied polyamines, but supplementation with as little as 10 ؊8 M spermidine restores their growth. Here, we report that culturing a spe1⌬ mutant or a spe2⌬ mutant in a standard polyamine-free minimal medium (SDC) leads to marked increases in cellular Mg 2؉ content. To determine which yeast Mg 2؉ transporter mediated this increase, we generated mutant strains with a deletion of SPE1 or SPE2 combined with a deletion of one of the three Mg 2؉ transporter genes, ALR1, ALR2, and MNR2, known to maintain cytosolic Mg 2؉ concentration. Neither Alr2 nor Mnr2 was required for increased Mg 2؉ accumulation, as all four double mutants (spe1⌬ alr2⌬, spe2⌬ alr2⌬, spe1⌬ mnr2⌬, and spe2⌬ mnr2⌬) exhibited significant Mg 2؉ accumulation upon polyamine depletion. In contrast, a spe2⌬ alr1⌬ double mutant cultured in SDC exhibited little increase in Mg 2؉ content and displayed severe growth defects compared with single mutants alr1⌬ and spe2⌬ under polyamine-deficient conditions. These findings indicate that Alr1 is required for the up-regulation of the Mg 2؉ content in polyamine-depleted cells and suggest that elevated Mg 2؉ can support growth of polyamine-deficient S. cerevisiae mutants. Upregulation of cellular polyamine content in a Mg 2؉-deficient alr1⌬ mutant provided further evidence for a cross-talk between Mg 2؉ and polyamine metabolism. The polyamines putrescine (NH 2 (CH 2) 4 NH 2), spermidine (NH 2 (CH 2) 3 NH(CH 2) 4 NH 2), and spermine (NH 2 (CH 2) 3 NH