The N terminus of the centromere H3-like protein Cse4p performs an essential function distinct from that of the histone fold domain

Cse4p is an evolutionarily conserved histone H3-like protein that is though t to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cer evisiae) centromere. All known yeast, worm, fly, and human centromere H3-li ke proteins have highly conserved C-terminal histone fold domains (HFD) but very different N tel mini. We have carried out a comprehensive and systema tic mutagenesis of the Cse4p N terminus to analyze its function. Surprising ly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus i s required for Cse4p N-terminal function. The spacing of the essential N-te rminal domain (END) relative to the HFD can be changed significantly withou t an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that C se4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These resu lts support different roles for the Cse4p N terminus and the HFD in centrom ere function and are consistent with the proposed Cse4p nucleosome model. T he structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP -A, function in kinetochore assembly and chromosome segregation.