Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123

Abstract

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kluyveromyces lactis Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.

Evidence for the nuclear import of histones H3.1 and H4 as monomers

“…mCherry on its own served as a control for non‐specific binding. Recent crystal structures have identified the importin‐β protein TNPO1 (KPNB2, MIP1), closely related to KPNB1 (O'Reilly et al , ), as interacting with the H3 tail (Fig EV4) (Soniat & Chook, ), and the Kluyveromyces lactis Kap123, a homolog of IPO4, as interacting with both H3 and H4 tails (An et al , ), building on previous biochemical and genetic analysis (Mosammaparast et al , ; Blackwell et al , ; Soniat & Chook, ).…”

“…Although studies have reported the role of lysine to glutamine mutations (as acetyl‐lysine mimics) in aiding nuclear import in P. polycephalum (Ejlassi‐Lassallette et al , ), a recent crystal structure has revealed the importance of unacetylated H4 tail peptides in mediating interaction with the K. lactis importin protein Kap123 (An et al , ). In addition, whilst crystal structures have revealed the importance of histone tail regions in binding a number of importin‐β proteins (Soniat & Chook, ; An et al , ), our truncation analysis would suggest that the junction with the core domain is of more importance in efficient nuclear delivery. This is supported by a recent study demonstrating that tail swaps result in lower rates of nuclear import (Ejlassi et al , ).…”

“…We probed a number of importin‐β proteins and found IPO4 and KPNB1 associated with cytosolically tethered H3.1 and/or H4, but do not accumulate in the nucleus upon histone release, suggesting a rapid hand‐off event to the nuclear chaperoning machinery. Interestingly, current evidence regarding the binding sites of importin‐β proteins suggests they interact with regions in the histone tails (Baake et al , ; Mosammaparast et al , ; Blackwell et al , ; Soniat & Chook, ; Soniat et al , ), with a recent crystal structure identifying the H3 epitope that binds to TNPO1 (Soniat & Chook, ) and H3 and H4 epitopes that bind to the yeast homolog Kap123 (An et al , ). Dumping of monomeric histones in the nucleus is unlikely; therefore, the molecular mechanism of how to transfer between importins and chaperone, and how this is coordinated with Ran‐GTP binding, may be of future interest.…”

Hat1 acetylates histone H4 and modulates the transcriptional program in Drosophila embryogenesis

“…The nuclear localization signals (NLS) of histones H3 and H4 can be found at their positively charged N-terminal tails 27 . Analysis of the crystal structure of the Kluyveromyces lactis IPO4 homologue (Kap123) revealed that the H3-NLS and H4-NLS are competing for Kap123 binding: the H3K14, H3K23 and H4K16 residues are recognized by lysine binding pockets of Kap123 28 . Lysine to glutamine mutations of H4K16 or H4K5/K12 reduced the affinity of H4 towards Kap123, suggesting that positive charge at these positions facilitates karyopherin binding 28 .…”

“…Analysis of the crystal structure of the Kluyveromyces lactis IPO4 homologue (Kap123) revealed that the H3-NLS and H4-NLS are competing for Kap123 binding: the H3K14, H3K23 and H4K16 residues are recognized by lysine binding pockets of Kap123 28 . Lysine to glutamine mutations of H4K16 or H4K5/K12 reduced the affinity of H4 towards Kap123, suggesting that positive charge at these positions facilitates karyopherin binding 28 . In Saccharomyces , mutation analysis of the H4 N-terminus in the absence of the H3-NLS showed that simultaneous mutations of H4 K5/K8/K12, and to a lesser extent, K5/K12 to glutamine resulted in cytoplasmic mislocalization of H4, while mutations to arginine did not have a similar effect 13 .…”