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Propagation of centromere identity during cell cycle progression in higher eukaryotes depends critically on the faithful incorporation of a centromere specific histone H3 variant encoded by CENPA in humans and cid in Drosophila. Cenp A/Cid is required for the recruitment of Cenp C another conserved centromere protein. With yeast three hybrid experiments we demonstrate that the essential Drosophila centromere protein Cal1 can link Cenp A/Cid and Cenp C. Cenp A/Cid and Cenp C interact with the N and C terminal domains of Cal1 respectively. These Cal1 domains are sufficient for centromere localization and function but only when linked together. Using quantitative in vivo imaging to determine protein copy numbers at centromeres and kinetochores we demonstrate that centromeric Cal1 levels are far lower than those of Cenp A/Cid Cenp C and other conserved kinetochore components which scale well with the number of kinetochore microtubules when comparing Drosophila with budding yeast. Rather than providing a stoichiometric link within the mitotic kinetochore Cal1 limits centromeric deposition of Cenp A/Cid and Cenp C during exit from mitosis. We demonstrate that the low amount of endogenous Cal1 prevents centromere expansion and mitotic kinetochore failure when Cenp A/Cid and Cenp C are present in excess.

The kinetochore is assembled during mitotic and meiotic divisions within the centromeric region of chromosomes. It is composed of more than eighty different proteins. Spc105 (also designated as Spc7 KNL 1 or Blinkin in different eukaryotes) is a comparatively large kinetochore protein which can bind to the Mis12/MIND and Ndc80 complexes and to the spindle assembly checkpoint components Bub1 and BubR1. Our genetic characterization of Drosophila Spc105 shows that a truncated version lacking the rapidly evolving repetitive central third still provides all essential functions. Moreover in comparison with Cenp C that has previously been observed to extend from the inner to the outer kinetochore region full length Spc105 is positioned further out and is not similarly extended along the spindle axis. Thus our results indicate that Spc105 forms neither an extended link connecting inner Cenp A chromatin with outer kinetochore regions nor a scaffold constraining kinetochore subcomplexes and spindle assembly checkpoint components together into a geometrically rigid supercomplex. Spc105 seems to provide a platform within the outer kinetochore allowing independent assembly of various kinetochore components.