Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast.

Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin.

Phosphorylation by casein kinase 2 facilitates Psh1 protein-assisted degradation of Cse4 protein.

Cse4 is the centromeric histone H3 variant in budding yeast. Psh1 is an E3 ubiquitin ligase that controls Cse4 levels through proteolysis. Here we report that Psh1 is phosphorylated by the Cka2 subunit of casein kinase 2 (CK2) to promote its E3 activity for Cse4. Deletion of CKA2 significantly stabilized Cse4. Consistent with phosphorylation promoting the activity of Psh1, Cse4 was stabilized in a Psh1 phosphodepleted mutant strain in which the major phosphorylation sites were changed to alanines. Phosphorylation of Psh1 did not control Psh1-Cse4 or Psh1-Ubc3(E2) interactions. Although Cse4 was highly stabilized in a cka2Δ strain, mislocalization of Cse4 was mild, suggesting that Cse4 misincorporation was prevented by the intact Psh1-Cse4 association. Supporting this idea, Psh1 was also stabilized in a cka2Δ strain. Collectively our data suggest that phosphorylation is crucial in Psh1-assisted control of Cse4 levels and that the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation.

Methods development for Analysis of Partially Deglycosylated Proteins and Application to an HIV Envelope Protein Vaccine Candidate.

The work presented herein describes the first comprehensive analysis of a partially deglycosylated HIV vaccine candidate envelope protein (Env). The Env, JRFL gp140 ΔCF, with 27 potential glycosylation sites, was partially deglycosylated with PNGase F as part of a strategy to generate a more immunogenic HIV vaccine, and the resulting protein's glycosylation was characterized in a unique workflow using two different glycosidases, Endo H and Endo F3. This unique analysis protocol provided for coverage on 26 of the 27 glycosylation sites, and the data showed that the biochemical treatment with PNGase F resulted in a highly heterogeneous glycoprotein product that had been partially deglycosylated at most of the glycosylation sites. The protocols described in this work could be useful for characterizing the glycosylation site occupancy of other native or biochemically deglycosylated proteins.

Characterization of glycosylation profiles of HIV-1 transmitted/founder envelopes by mass spectrometry.

The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simplification of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workflow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan profiles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades.

Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4.

Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.