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Chromosome biorientation is promoted by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore-microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore, and spindle microtubules. Here we show that a small domain of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This domain, the single alpha helix (SAH), is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of its three locations through targeted mutations and fusion constructs, we determined their individual contributions to chromosome biorientation. We find that only the inner centromere localization is sufficient for cell viability on its own. However, when combined, the inner kinetochore and microtubule binding activities are also sufficient to promote accurate chromosome segregation. Furthermore, we find that the two pathways target the CPC to different kinetochore attachment states, as the inner centromere-targeting pathway is primarily responsible for bringing the complex to unattached kinetochores. We have therefore discovered that two parallel localization pathways are each sufficient to promote CPC activity in chromosome biorientation, both depending on the SAH domain of INCENP/Sli15.

Chromosome biorientation is established by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore-microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore and spindle microtubules. Here we show that a small region of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This region, the SAH, is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of these three locations individually, we found that inner centromere localization is sufficient to establish chromosome biorientation and viability independently of the other two targeting mechanisms. Remarkably, although neither the inner kinetochore nor microtubule binding activities was able to rescue viability individually, they were able to do so when combined. We have therefore identified two parallel pathways by which the CPC can promote chromosome biorientation and proper completion of mitosis.