Yeast Nap1-binding protein Nbp2p is required for mitotic growth at high temperatures and for cell wall integrity

Nbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid system. NBP2 encodes a novel protein consisting of 236 amino acids with a Src homology 3 (SH3) domain. We showed that NBP2 functions to promote mitotic cell growth at high temperatures and cell wall integrity. Loss of Nbp2 results in cell death at high temperatures and in sensitivity to calcofluor white. Cell death at high temperature is thought not to be due to a weakened cell wall. Additionally, we have isolated several type-2C serine threonine protein phosphatases (PTCs) as multicopy suppressors and MAP kinase-kinase (MAPKK), related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2Delta mutant. Screening for deletion suppressors is a new genetic approach to identify and characterize additional proteins in the Nbp2-dependent pathway. Genetic analyses suggested that Ptc1, which interacts with Nbp2 by the two-hybrid system, acts downstream of Nbp2 and that cells lacking the function of Nbp2 prefer to lose Mkk1, but the PKC MAPK pathway itself is indispensable when Nbp2 is deleted at high temperature.     

Involvement of nucleocytoplasmic shuttling of yeast Nap1 in mitotic progression

Nucleosome assembly protein 1 (Nap1) is widely conserved from yeasts to humans and facilitates nucleosome formation in vitro as a histone chaperone. Nap1 is generally localized in the cytoplasm, except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development. The cytoplasmic localization of Nap1 is seemingly incompatible with the proposed role of Nap1 in nucleosome formation, which should occur in the nucleus. Here, we have examined the roles of a putative nuclear export signal (NES) sequence in yeast Nap1 (yNap1). yNap1 mutants lacking the NES-like sequence were localized predominantly in the nucleus. Deletion of NAP1 in cells harboring a single mitotic cyclin gene is known to cause mitotic delay and temperature-sensitive growth. A wild-type NAP1 complemented these phenotypes while nap1 mutant genes lacking the NES-like sequence or carboxy-terminal region did not. These and other results suggest that yNap1 is a nucleocytoplasmic shuttling protein and that its shuttling is important for yNap1 function during mitotic progression. This study also provides a possible explanation for Nap1's involvement in nucleosome assembly and/or remodeling in the nucleus.     

A role for nucleosome assembly protein 1 in the nuclear transport of histones H2A and H2B

Import of core histones into the nucleus is a prerequisite for their deposition onto DNA and the assembly of chromatin. Here we demonstrate that nucleosome assembly protein 1 (Nap1p), a protein previously implicated in the deposition of histones H2A and H2B, is also involved in the transport of these two histones. We demonstrate that Nap1p can bind directly to Kap114p, the primary karyopherin/importin responsible for the nuclear import of H2A and H2B. Nap1p also serves as a bridge between Kap114p and the histone nuclear localization sequence (NLS). Nap1p acts cooperatively to increase the affinity of Kap114p for these NLSs. Nuclear accumulation of histone NLS-green fluorescent protein (GFP) reporters was decreased in deltanap1 cells. Furthermore, we demonstrate that Nap1p promotes the association of the H2A and H2B NLSs specifically with the karyopherin Kap114p. Localization studies demonstrate that Nap1p is a nucleocytoplasmic shuttling protein, and genetic experiments suggest that its shuttling is important for maintaining chromatin structure in vivo. We propose a model in which Nap1p links the nuclear transport of H2A and H2B to chromatin assembly.     

Phosphorylation by casein kinase 2 regulates Nap1 localization and function

In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.     

Distinct roles for histone chaperones in the deposition of Htz1 in chromatin

Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4)₂ tetramers, H2A–H2B dimers and Htz1–H2B dimers. Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin.     

TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1

Certain proteins can undergo polyglycylation and polyglutamylation. Polyglutamylases (glutamate ligases) have recently been identified in a family of tubulin tyrosine ligase-like (TTLL) proteins. However, no polyglycylase (glycine ligase) has yet been reported. Here we identify a polyglycylase in the TTLL proteins by using an anti-poly-glycine antibody. The antibody reacted with a cytoplasmic 60-kDa protein that accumulated in elongating spermatids. Using tandem mass spectrometry of trypsinized samples, immunoprecipitated by the antibody from the TTLL10-expressing cells, we identified the 60-kDa protein as nucleosome assembly protein 1 (NAP1). Recombinant TTLL10 incorporated glycine into recombinant NAP1 in vitro. Mutational analyses indicated that Glu residues at 359 and 360 in the C-terminal part of NAP1 are putative sites for the modification. Thus, TTLL10 is a polyglycylase for NAP1.     

Analysis of wild-type and mutant p21WAF-1 gene activities.

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.     

Human papillomavirus E1 helicase interacts with the WD repeat protein p80 to promote maintenance of the viral genome in keratinocytes

Due to the limited coding capacity of their small genomes, human papillomaviruses (HPV) rely extensively on host factors for the completion of their life cycles. Accordingly, most HPV proteins, including the replicative helicase E1, engage in multiple protein interactions. The fact that conserved regions of E1 have not yet been ascribed a function prompted us to use tandem affinity protein purification (TAP) coupled to mass spectrometry to identify novel targets of this helicase. This method led to the discovery of a novel interaction between the N-terminal 40 amino acids of HPV type 11 (HPV11) E1 and the cellular WD repeat protein p80 (WDR48). We found that interaction with p80 is conserved among E1 proteins from anogenital HPV but not among cutaneous or animal types. Colocalization studies showed that E1 can redistribute p80 from the cytoplasm to the nucleus in a manner that is dependent on the E1 nuclear localization signal. Three amino acid substitutions in E1 proteins from HPV11 and -31 were identified that abrogate binding to p80 and its relocalization to the nucleus. In HPV31 E1, these substitutions reduced but did not completely abolish transient viral DNA replication. HPV31 genomes encoding two of the mutant E1 proteins were not maintained as episomes in immortalized primary keratinocytes, whereas one encoding the third mutant protein was maintained at a very low copy number. These findings suggest that the interaction of E1 with p80 is required for efficient maintenance of the viral episome in undifferentiated keratinocytes.     

The synergy between RSC,Nap1 and adjacent nucleosome in nucleosome remodeling

Eukaryotes have evolved a specific strategy to package DNA. The nucleosome is a 147-base-pair DNA segment wrapped around histone core proteins that plays important roles regulating DNA-dependent biosynthesis and gene expression. Chromatin remodeling complexes (RSC, Remodel the Structure of Chromatin) hydrolyze ATP to perturb DNA-histone contacts, leading to nucleosome sliding and ejection. Here, we utilized tethered particle motion (TPM) experiments to investigate the mechanism of RSC-mediated nucleosome remodeling in detail. We observed ATP-dependent RSC-mediated DNA looping and nucleosome ejection along individual mononucleosomes and dinucleosomes. We found that nucleosome assembly protein 1 (Nap1) enhanced RSC-mediated nucleosome ejection in a two-step disassembly manner from dinucleosomes but not from mononucleosomes. Based on this work, we provide an entire reaction scheme for the RSC-mediated nucleosome remodeling process that includes DNA looping, nucleosome ejection, the influence of adjacent nucleosomes, and the coordinated action between Nap1 and RSC.