Tony R. Hazbun

Assigning Function to Yeast Proteins by Integration of Technologies

Interpreting genome sequences requires the functional analysis of thousands of predicted proteins, many of which are uncharacterized and without obvious homologs. To assess whether the roles of large sets of uncharacterized genes can be assigned by targeted application of a suite of technologies, we used four complementary protein-based methods to analyze a set of 100 uncharacterized but essential open reading frames (ORFs) of the yeast Saccharomyces cerevisiae. These proteins were subjected to affinity purification and mass spectrometry analysis to identify copurifying proteins, two-hybrid analysis to identify interacting proteins, fluorescence microscopy to localize the proteins, and structure prediction methodology to predict structural domains or identify remote homologies. Integration of the data assigned function to 48 ORFs using at least two of the Gene Ontology (GO) categories of biological process, molecular function, and cellular component; 77 ORFs were annotated by at least one method. This combination of technologies, coupled with annotation using GO, is a powerful approach to classifying genes.

A Novel, Evolutionarily Conserved Protein Phosphatase Complex Involved in Cisplatin Sensitivity

Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2).

Networking proteins in yeast

The advent of genome sequencing projects—culminating in the recent reports of the human sequence (1, 2)—has resulted in both the identification of novel genes and proteins as well as the proliferation of the “omes” that come from their analyses: the proteome (the complement of proteins), transcriptome (the complement of mRNA transcripts), metabolome (the complement of metabolites), and so on. These end products of global assays are needed to interpret the large fraction (typically close to half) of predicted proteins for which no proteins of similar structure exist or have been functionally characterized. The report by Ito et al. (3) is the largest contribution to date in the effort to generate the protein interactome, or map of protein–protein interactions, for the yeast Saccharomyces cerevisiae.

Sumoylation of the budding yeast kinetochore protein Ndc10 is required for Ndc10 spindle localization and regulation of anaphase spindle elongation

Posttranslational modification by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) is emerging as an important regulator in many cellular processes, including genome integrity. In this study, we show that the kinetochore proteins Ndc10, Bir1, Ndc80, and Cep3, which mediate the attachment of chromosomes to spindle microtubules, are sumoylated substrates in budding yeast. Furthermore, we show that Ndc10, Bir1, and Cep3 but not Ndc80 are desumoylated upon exposure to nocodazole, highlighting the possibility of distinct roles for sumoylation in modulating kinetochore protein function and of a potential link between the sumoylation of kinetochore proteins and mitotic checkpoint function. We find that lysine to arginine mutations that eliminate the sumoylation of Ndc10 cause chromosome instability, mislocalization of Ndc10 from the mitotic spindle, abnormal anaphase spindles, and a loss of Bir1 sumoylation. These data suggest that sumoylation of Ndc10 and other kinetochore proteins play a critical role during the mitotic process.

Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore–spindle binding and chromosome segregation are mediated by the multi-copy KNL1Spc105, MIS12Mtw1 and NDC80Ndc80xa0complexes that form the so-called KMN network. KMN–spindle attachment is regulated by the Auroraxa0BIpl1 and MPS1Mps1xa0kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.

A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications

We have modified the yeast two-hybrid system to enable the detection of protein-protein interactions that require a specific post-translational modification, using the acetylation of histones and the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II as test modifications. In this tethered catalysis assay, constitutive modification of the protein to be screened for interactions is achieved by fusing it to its cognate modifying enzyme, with the physical linkage resulting in efficient catalysis. This catalysis maintains substrate modification even in the presence of antagonizing enzyme activities. A catalytically inactive mutant of the enzyme is fused to the substrate as a control such that the modification does not occur; this construct enables the rapid identification of modification-independent interactions. We identified proteins with links to chromatin functions that interact with acetylated histones, and proteins that participate in RNA polymerase II functions and in CTD phosphorylation regulation that interact preferentially with the phosphorylated CTD.

A pooling-deconvolution strategy for biological network elucidation

The generation of large-scale data sets is a fundamental requirement of systems biology. But despite recent advances, generation of such high-coverage data remains a major challenge. We developed a pooling-deconvolution strategy that can dramatically decrease the effort required. This strategy, pooling with imaginary tags followed by deconvolution (PI-deconvolution), allows the screening of 2n probe proteins (baits) in 2 × n pools, with n replicates for each bait. Deconvolution of baits with their binding partners (preys) can be achieved by reading the preys profile from the 2 × n experiments. We validated this strategy for protein-protein interaction mapping using both proteome microarrays and a yeast two-hybrid array, demonstrating that PI-deconvolution can be used to identify interactions accurately with fewer experiments and better coverage. We also show that PI-deconvolution can be used to identify protein-small molecule interactions inferred from profiling the yeast deletion collection. PI-deconvolution should be applicable to a wide range of library-against-library approaches and can also be used to optimize array designs.

Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

Plk1 phosphorylation of Orc2 and Hbo1 contributes to gemcitabine resistance in pancreatic cancer

Although gemcitabine is the standard chemotherapeutic drug for treatment of pancreatic cancer, almost all patients eventually develop resistance to this agent. Previous studies identified Polo-like kinase 1 (Plk1) as the mediator of gemcitabine resistance, but the molecular mechanism remains unknown. In this study, we show that Plk1 phosphorylation of Orc2 and Hbo1 mediates the resistance to gemcitabine. We show that the level of Plk1 expression positively correlates with gemcitabine resistance, both in pancreatic cancer cells and xenograft tumors. Overexpression of Plk1 increases gemcitabine resistance, while inhibition of Plk1 sensitizes pancreatic cancer cells to gemcitabine treatment. To validate our findings, we show that inhibition of Plk1 sensitizes tumors to gemcitabine treatment in a mouse xenograft study. Mechanistically, we find that Plk1 phosphorylation of Orc2 maintains DNA replication on gemcitabine treatment. Furthermore, Plk1 phosphorylation of Hbo1 transcriptionally increases cFos expression and consequently elevates its target multidrug resistance 1 (MDR1), which was previously reported to confer chemotherapeutic drug resistance. Knockdown of cFos or MDR1 sensitizes gemcitabine-resistant cells to gemcitabine treatment. Finally, pancreatic cancer cells expressing Plk1-unphosphorylatable mutants of Orc2 or Hbo1 are more sensitive to gemcitabine than cells expressing wild-type Orc2 or Hbo1. In short, our study provides a mechanism for Plk1-mediated gemcitabine resistance, suggesting that Plk1 is a promising target for treatment of gemcitabine-resistant pancreatic cancer. Mol Cancer Ther; 12(1); 58–68. ©2012 AACR.

Histone H3 exerts a key function in mitotic checkpoint control

ABSTRACT It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of Sgo1p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint.