Characterization of the Multifunctional Histone Chaperones Nap1 and Vps75

Histone chaperones of the Nap1 family are evolutionarily conserved, multifunctional proteins whose misregulation in higher eukaryotes is correlated with disease. Saccharomyces cerevisiae has two members of this family, Nap1, a nucleocytoplasmic shuttling protein known to promote H2A/H2B import, and Vps75, a recently characterized H3/H4 chaperone that affects H3 acetylation. Besides their histone chaperone activities, Vps75 associates with and affects the activity of a histone acetyltransferase and Nap1 is involved in regulation of mitotic progression. Histone modification is critical for the regulation of all chromatin-templated processes. Vps75 associates with and stabilizes Rtt109, the histone acetyltransferase for H3 lysines 9, 27 and 56, but the effect of Vps75 on Rtt109 activity in vivo was largely unknown. We determined that Vps75 nuclear localization is mediated by a classical NLS and Kap60-Kap95 and promotes Rtt109 nuclear localization. Nuclear localization of the Vps75-Rtt109 complex is not critical for Rtt109-dependent functions, suggesting Rtt109 may acetylate newly synthesized histones before nuclear import. To understand Vps75 function, we used an Rtt109 mutant lacking the Vps75 interaction domain that is stable without Vps75. The Rtt109-Vps75 interaction was essential for H3 K27 acetylation and full K9 acetylation. Our results suggest that Vps75 brings together the HAT and histones and allows Rtt109 access to the H3 tail. Furthermore, our genetic interaction data point to Rtt109-independent functions of Vps75. The timing of mitotic entry must be coordinated with daughter bud formation to produce viable progeny in yeast. Many of the proteins that regulate mitotic entry, such as Nap1 and the Nim1-related kinases: Hsl1, Gin4 and Kcc4, localize to the motheriii daughter bud neck where they can monitor bud formation and impose a G2/M delay when necessary. We hypothesized that the mitotic functions of Nap1 may be regulated by its physical interactions and by phosphorylation. We identified Nap1 interacting proteins and Nap1 phosphorylated residues. Our data indicate that Nap1 interacts with all three Nim1-related kinases in vivo and we explored the possibility that one of these kinases, Hsl1, may regulate Nap1 functions through phosphorylation. Furthermore, we show that Nap1 and its interacting protein Nba1 are phosphorylated by Cdc28-Clb2. We believe that the pleiotropic functions of Nap1 may serve to integrate into one protein a variety of signals from different phases of the cell cycle. Nap1 and its homolog Vps75 are histone chaperones with functions in a variety of cellular processes such as chromatin assembly, histone transport, histone posttranslational modification, cell cycle regulation and transcription.

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