SUMMARY 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.

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.

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.

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.

A number of the Saccharomyces cerevisiae vacuolar protein-sorting (vps) mutants exhibit an altered vacuolar morphology. Unlike wild-type cells that contain 1-3 large vacuolar structures, the class B vps5 and vpsl7 mutant cells contain 10-20 smaller vacuole-like compartments. To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its protein product. The VPS5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that shows significant sequence homology with mammalian sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic protein that is phosphorylated specifically on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and vacuolar membranes. This association was found to be dependent on the presence of another class B VPS gene product, Vpsl7p. Biochemical cross-linking studies demonstrated that Vps5p and Vpsl7p physically interact. Gene disruption experiments show that the VPS5 gene product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in vacuolar proteinsorting similar to vpsl7 null mutants. More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form. Additionally, the VpslOp vacuolar protein-sorting receptor is mislocalized to the vacuole in vps5 mutant cells. On the basis of these and other observations, we propose that the Vps5p/Vpsl7p protein complex may participate in the intracellular trafficking of the VpslOp-sorting receptor, as well as other late-Golgi proteins.