David T. Stuart

Structural elucidation of a PRP8 core domain from the heart of the spliceosome

The spliceosome is a complex ribonucleoprotein (RNP) particle containing five RNAs and more than 100 associated proteins. One of these proteins, PRP8, has been shown to interact directly with the splice sites and branch region of precursor-mRNAs (pre-mRNAs) and spliceosomal RNAs associated with catalysis of the two steps of splicing. The 1.85-Å X-ray structure of the core of PRP8 domain IV, implicated in key spliceosomal interactions, reveals a bipartite structure that includes the presence of an RNase H fold linked to a five-helix assembly. Analysis of mutant yeast alleles and cross-linking results in the context of this structure, coupled with RNA binding studies, suggests that domain IV forms a surface that interacts directly with the RNA structures at the catalytic core of the spliceosome.

Structure of the sporulation-specific transcription factor Ndt80 bound to DNA

Progression through the middle phase of sporulation in Saccharomyces cerevisiae is promoted by the successful completion of recombination at the end of prophase I. Completion of meiotic recombination allows the activation of the sporulation‐specific transcription factor Ndt80, which binds to a specific DNA sequence, the middle sporulation element (MSE), and activates ∼150 genes to enable progression through meiosis. Here, we isolate the DNA‐binding domain of Ndt80 and determine its crystal structure both free and in complex with an MSE‐containing DNA. The structure reveals that Ndt80 is a member of the Ig‐fold family of transcription factors. The structure of the DNA‐bound form, refined at 1.4 Å, reveals an unexpected mode of recognition of 5′‐pyrimidine–guanine‐3′ dinucleotide steps by arginine residues that simultaneously recognize the 3′‐guanine base through hydrogen bond interactions and the 5′‐pyrimidine through stacking/van der Waals interactions. Analysis of the DNA‐binding affinities of MSE mutants demonstrates the central importance of these interactions, and of the AT‐rich portion of the MSE. Functional similarities between Ndt80 and the Caenorhabditis elegans p53 homolog suggest an evolutionary link between Ndt80 and the p53 family.

Phosphorylation and maximal activity of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is dependent on Ime2.

ABSTRACT The Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is responsible for the induction of a class of genes referred to as middle sporulation genes. Among the members of this family are the B-type cyclins and other genes whose products are required for meiotic chromosome division and spore morphogenesis. Inactivation of NDT80 leads to a failure to induce the middle sporulation genes and a subsequent arrest in pachytene. The expression of NDT80 is itself highly regulated. The initial transcription of NDT80 is dependent upon the protein kinase Ime2; once Ndt80 protein accumulates, it activates its own promoter, thus generating an autoactivation loop. In addition to being transcriptionally regulated, Ndt80 protein is posttranslationally regulated. Phosphorylation of Ndt80 occurs coincident with its activation as a transcription factor. If expressed prematurely in meiosis, Ndt80 accumulates initially in an unmodified form that is subsequently modified by phosphorylation. In contrast, Ndt80 expressed in ime2 mutant strains does not become modified and has a reduced ability to activate transcription of its target genes. Ime2 can also phosphorylate Ndt80 in vitro, further supporting a direct role for Ime2 in the phosphorylation of Ndt80. These data indicate that Ime2 plays a novel and previously unexpected role in promoting chromosome dissemination and progress through meiotic development by activating Ndt80.

Saccharomyces cerevisiae Ime2 phosphorylates Sic1 at multiple PXS/T sites but is insufficient to trigger Sic1 degradation

The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb-Cdc28 inhibitor Sic1. In proliferating cells Cln-Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCF(Cdc4) and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCF(Cdc4) was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCF(Cdc4). In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.

Analyses of Phosphorylation Events in the Rubella Virus Capsid Protein: Role in Early Replication Events

ABSTRACT The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA binding region of the capsid is required to trigger phosphorylation of additional amino acid residues that include threonine 47. This residue likely plays a direct role in regulating the binding of genomic RNA to the capsid. We also provide evidence which suggests that the capsid is dephosphorylated prior to or during virus budding. Finally, whereas the phosphorylation state of the capsid does not directly influence the rate of synthesis of viral RNA and proteins or the assembly and secretion of virions, the presence of phosphate on the capsid is critical for early events in virus replication, most likely the uncoating of virions and/or disassembly of nucleocapsids.

Dual Phosphorylation of Cdk1 Coordinates Cell Proliferation with Key Developmental Processes in Drosophila

Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Here, we expressed wild-type and phospho-acceptor mutant Cdk1 proteins to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2-phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the premitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1–Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2-phase cells are ready for mitosis.

Engineering Saccharomyces cerevisiae fermentative pathways for the production of isobutanol

Background: Finite supplies of petroleum-based fossil fuels, in addition to concerns about carbon emissions and energy security, have driven the search for alternative fuels that can be produced from renewable resources. Butanol, pentanol and their isomers have significant advantages over ethanol as a biofuel and these can be produced by fermentation. Results: We demonstrate that yeast can be engineered to produce isobutanol by fermentation of carbohydrate precursors. This was achieved by increasing flux through the valine biosynthetic pathway in addition to decreasing pyruvate decarboxylase activity and increasing the availability of NADPH. We found no initial improvement in isobutanol production by deleting BAT1, LEU4 and LEU9, genes encoding enzymes predicted to compete with isobutanol synthesis. Conclusion: Yeast has potential as a factory for the production of higher alcohol biofuels; however, substantial engineering will be required to achieve economically viable production levels.

The Cdc34/SCF ubiquitination complex mediates Saccharomyces cerevisiae cell wall integrity.

To identify novel functions for the Cdc34/SCF ubiquitination complex, we analyzed genomewide transcriptional profiles of cdc53-1 and cdc34-2 Saccharomyces cerevisiae mutants. This analysis revealed altered expression for several gene families, including genes involved in the regulation of cell wall organization and biosynthesis. This led us to uncover a role for the Cdc34/SCF complex in the regulation of cell wall integrity. In support of this, cdc53-1 and cdc34-2 mutants exhibit phenotypes characteristic of cell wall integrity mutants, such as SDS sensitivity and temperature-sensitive suppression by osmotic stabilizers. Examination of these mutants revealed defects in their induction of Slt2 phosphorylation, indicating defects in Pkc1-Slt2 MAPK signaling. Consistent with this, synthetic genetic interactions were observed between the genes encoding the Cdc34/SCF complex and key components of the Pck1-Slt2 MAPK pathway. Further analysis revealed that Cdc34/SCF mutants have reduced levels of active Rho1, suggesting that these defects stem from the deregulated activity of the Rho1 GTPase. Altering the activity of Rho1 via manipulation of the Rho1-GAPs LRG1 or SAC7 affected Cdc34/SCF mutant growth. Strikingly, however, deletion of LRG1 rescued the growth defects associated with Cdc34/SCF mutants, whereas deletion of SAC7 enhanced these defects. Given the differential roles that these GAPs play in the regulation of Rho1, these observations indicate the importance of coordinating Cdc34/SCF activity with specific Rho1 functions.

Purification and some properties of Saccharomyces cerevisiae meiosis-specific protein kinase Ime2

Ime2 is the founding member of a family of protein kinases that are required for effective progression through meiotic development. Ime2 is essential for the induction of meiosis-specific genes and for the activation of meiotic DNA replication in the budding yeast Saccharomyces cerevisiae. Aside from the fact that Ime2 is a protein kinase and shares several amino acid motifs with cyclin dependent kinases, virtually nothing is known about its enzymatic properties or substrates. Biochemical characterization of Ime2 has been hindered by its low abundance and short half-life. We have created baculovirus expression vectors to produce recombinant Ime2 in insect cells. In this report, we describe the overproduction of Ime2 and its purification using affinity chromatography. Using this procedure, we have been able to purify up to 2mg Ime2 from 1L of infected insect cells. The Ime2 isolated by this method displays properties similar to those of the native enzyme that has been immunoprecipitated from yeast. The high level expression of Ime2 in this system and its ease of purification will be beneficial for more extensive biochemical analysis of Ime2 and related meiosis-specific kinases.

Among B-Type Cyclins Only CLB5 and CLB6 Promote Premeiotic S Phase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cyclin Clb5 is required for premeiotic S phase, meiotic recombination, and successful progression through meiosis. Clb5 is not essential for mitotic proliferation because Clb1–Clb4 can support DNA replication in clb5 clb6 mutants. Clb1, Clb3, and Clb4 accumulate in clb5 clb6 cells during meiotic differentiation yet fail to promote premeiotic DNA replication. When expressed under the regulation of the CLB5 promoter, Clb1 and Clb3 accumulate and are active in the early stages of meiotic differentiation but cannot induce premeiotic DNA replication, suggesting that they do not target Cdk1 to the necessary substrates. The Clb5 hydrophobic patch (HP) residues are important for Clb5 function but this motif alone does not provide the specificity required for Clb5 to induce premeiotic S phase. Domain exchange experiments demonstrated that the amino terminus of Clb5 when fused to Clb3 confers upon Clb3 the ability to induce premeiotic S phase. Chimeric cyclins containing smaller regions of the Clb5 amino terminus displayed reduced ability to activate premeiotic DNA replication despite being more abundant and having greater associated histone H1 kinase activity than endogenous Clb5. These observations suggest that Clb5 has a unique ability to trigger premeiotic S phase and that the amino-terminal region of Clb5 contributes to its specificity and regulates the functions performed by the cyclin–Cdk complex.