Brian A. Davies

Acetylation of Histone H3 Lysine 56 Regulates Replication-Coupled Nucleosome Assembly

Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106.

Mechanism of ubiquitin recognition by the CUE domain of Vps9p.

Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways. The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain. The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule. The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin. The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule. Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin.

Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1

In eukaryotes, the multivesicular body (MVB) sorting pathway plays an essential role in regulating cell surface protein composition, thereby impacting numerous cellular functions. Vps4, an ATPase associated with a variety of cellular activities, is required late in the MVB sorting reaction to dissociate the endosomal sorting complex required for transport (ESCRT), a requisite for proper function of this pathway. However, regulation of Vps4 function is not understood. We characterize Vta1 as a positive regulator of Vps4 both in vivo and in vitro. Vta1 promotes proper assembly of Vps4 and stimulates its ATPase activity through the conserved Vta1/SBP1/LIP5 region present in Vta1 homologues across evolution, including human SBP1 and Arabidopsis thaliana LIP5. These results suggest an evolutionarily conserved mechanism through which the disassembly of the ESCRT proteins, and thereby MVB sorting, is regulated by the Vta1/SBP1/LIP5 proteins.

ESCRT-III Family Members Stimulate Vps4 ATPase Activity Directly or via Vta1

The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.

Structural Basis of Vta1 Function in the Multivesicular Body Sorting Pathway

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.

Assembly of the AAA ATPase Vps4 on ESCRT-III

A complex network of interactions mediates the recruitment of Vps4 to ESCRT-III and its subsequent assembly, two key steps in the ESCRT-dependent vesicle formation at the endosome. A model is presented depicting the order of events that lead to active, ESCRT-III–associated Vps4.

Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106.

Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3–H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3–H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3–H4. An N-terminal domain homodimerizes and interacts with H3–H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3–H4 components of the (H3–H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3–H4)2. We show that the Rtt106–(H3–H4)2 interaction is important for gene silencing and the DNA damage response.

Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast

The ubiquitin hydrolase activating factor Bro1 enhances ESCRT-III stability by inhibiting Vps4-mediated disassembly.

Structural Basis of Ist1 Function and Ist1–Did2 Interaction in the Multivesicular Body Pathway and Cytokinesis

The ESCRT machinery functions in several important eukaryotic cellular processes. The AAA-ATPase Vps4 catalyzes disassembly of the ESCRT-III complex and may regulate membrane deformation and vesicle scission as well. Ist1 was proposed to be a regulator of Vps4, but its mechanism of action was unclear. The crystal structure of the N-terminal domain of Ist1 (Ist1NTD) reveals an ESCRT-III subunit-like fold, implicating Ist1 as a divergent ESCRT-III family member. Ist1NTD specifically binds to the ESCRT-III subunit Did2, and cocrystallization of Ist1NTD with a Did2 fragment shows that Ist1 interacts with the Did2 C-terminal MIM1 (MIT-interacting motif 1) via a novel MIM-binding structural motif. This arrangement indicates a mechanism for intermolecular ESCRT-III subunit association and may also suggest one form of ESCRT-III subunit autoinhibition via intramolecular interaction.

Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly

Vps4 disassembly of ESCRT-III plays an important role in MVB sorting, viral budding, and cytokinesis. An in vitro system was developed to investigate this process. These studies revealed new insights into the mechanisms of Vps4 function.