Histone variant incorporation into nucleosomes is a key mechanism for regulatingessential transactions with DNA, and diversifies chromatin structure and function.H2A.Z is an enigmatic H2A variant that is conserved across eukaryotes and hasdiverse roles in gene regulation, genome stability, chromosome segregation andDNA repair. The acid-patch containing M6 region of H2A.Z is essential for viabilityin Drosophila and imparts unique functions on H2A.Z in S. cerevisiae. However,many other structural determinants of H2A.Z function remain to be discovered.Novel point mutations I109T and S111P, in the H2A.Z C-terminal region, have beencharacterised here in S. cerevisiae, and show that important structural determinantsof H2A.Z cellular and gene regulatory functions reside outside of the M6 region.These mutations impair the resistance of cells to cytotoxic stress and causetranscriptional misregulation phenotypes, which correlate with defective chromatinassociation. Importantly, the loss of mutant H2A.Z from chromatin involves alteredinteractions with both of the SWR and INO80 chromatin remodelling complexes,and reduced physical stability within chromatin. Computational modelling of thesemutations in the human H2A.Z nucleosome structure predicts rearrangements inlocal structure, suggesting that the integrity of the C-terminal domain at thesepositions is a key determinant of H2A.Z function and chromatin association, throughmultiple mechanisms.