High throughput proteome-wide search for ubiquitinated proteins in yeast
Our goal is to improve upon existing biochemical approaches for finding sites of ubiquitin attachment on all yeast proteins.
Presently, the approach is to purify proteins with an 8xHis-tagged ubquitin followed by tryptic digest to generate peptide fragments for LC/LC-MS/MS analysis. The tryptic fragments that contain a lysine residue that had been ubiquitinated can be identified by a mass shift corresponding to 2 glycine residues left attached to an internal lysine. Historically, only a relatively small number of GG-peptides have been found due to low abundance of GG-peptide as compared to unmodified peptides.
We seek to reduce the complexity of the resulting peptides by using the chemical NTCB (2-nitro-5-thiocyanatobenzoic acid) which cleaves at cysteine residues (Figure 1B). Since ubiquitin does not have any cysteines, the proteome can be cleaved prior to affinity purification ubiquitinated proteins (Figure 1C) and typsinization (Figure 1D). This step will remove many non-ubiquitinated fragments therefore reducing the complexity of the sample. We hope to further decrease the complexity of the peptides by separating forked peptides from linear peptides by strong-cation exchange chromatography (Figure 1E) prior to injection into the mass spectrometer.
Using the technique outlined above we found a total of 965 unique sites of ubiquitin attachment on 410 yeast proteins. We are able to gain a 1.5X enrichment of GG-peptides using the NTCB cleavage strategy. Most of the improvement in sequencing GG-peptides is achieved by using the high-mass accuracy Orbitrap-LTQ (Table 1).
•Lea Starita & Russell Lo
Starita, L.M., Lo, R.S., Eng, J.K., Von Haller, P.D. and Fields, S. Sites of ubiquitin attachment in Saccharomyces cerevisiae. 2012 Proteomics, Jan;12(2):236-240. Epub 2011 Nov 22 download pdf
Genome-wide analysis of nascent transcription in Saccharomyces cerevisiae
Most studies of eukaryotic gene regulation have examined mature, steady-state mRNA levels. However, steady-state mRNA levels result from the action of two opposing processes: RNA synthesis and RNA degradation. An accurate assessment of RNA synthesis is important for understanding the mechanisms that regulate gene expression.
The nuclear run-on (NRO) assay is the traditional method to directly measure RNA synthesis. We have combined the in vivo RNA labeling of this assay with high throughput DNA sequencing to examine RNA polymerase activity genome-wide in exponentially growing yeast. In parallel, we sequenced total RNA to monitor transcript abundance and compare nascent transcript and steady-state transcript levels (Figure 1A).
To analyze RNA polymerase activity within genes, we examined read density along transcribed regions. We find that in contrast to total RNA libraries, NRO libraries show a high density of reads near the 5’ ends of the transcript models, with a peak ~50 bp downstream of the transcription start site (TSS) (Figure 1B), as has been observed in human and Drosophila cells. This peak in read depth near TSSs likely indicates a promoter-proximal accumulation of paused RNA polymerase, suggesting that pausing plays a significant role in the regulation of yeast transcription. Analysis of expression levels allows us to classify genes into four classes by their activity and pausing (Figure 1C). Ranking genes by the significance of pausing reveals that histone genes are among the 5% most paused genes, suggesting that transition to productive elongation is necessary for rapid induction of histone synthesis in S phase. By calculating the ratio of NRO transcription to total RNA for each gene, we can estimate nascent transcript stabilities. This analysis has revealed that the most stable and unstable transcripts encode proteins whose functional roles are consistent with these stabilities.
Parallel analysis of nascent transcripts and steady-state transcripts with high throughput sequencing allows a genome-wide assessment of RNA polymerase activity in yeast, identifying regulatory steps of RNA synthesis and inference of RNA stabilities. We anticipate that this approach will be useful to measure changes that occur in transcription in response to environmental or genetic perturbations.
McKinlay, A., Araya, C.L. and Fields, S. Genome-wide analysis of nascent transcription in Saccharomyces cerevisiae. 2011 G3: Genes, Genomes, Genetics Dec;1(7):549-58. Epub 2011 Dec 1. download pdf
•Anastasia McKinlay & Carlos Araya (former lab members)
An Integrated Metabolomic Approach to Understanding Drug Function
Metabolites are a unique and highly diverse class of elements and compounds that constitute the “business end” of biochemistry. For example, the budding yeast S. cerevisiae is estimated to contain thousands of unique metabolites at a wide range of concentrations. In addition to being both substrates for and products of protein action, metabolites have profound regulatory effects ranging from simple enzymatic product inhibition to allostery to initiation of complex signaling cascades that regulate gene expression programs. Furthermore, exogenous metabolites, acquired for nutritive purposes or used as chemical defenses, greatly expand the diversity of metabolites a cell might encounter. Thus, we hypothesized that examination of the effect of excess metabolites on a drug phenotype could provide rich, systems-level information about both cellular and drug function.
To test this principle, we screened a small pilot library of about 50 metabolites in a yeast-based model against lovastatin. Statin drugs inhibit HMG-CoA reductase, which is the rate-limiting enzyme in the synthesis of cholesterol. Consequently, they are among the most widely prescribed drugs in the world, used to treat high cholesterol and atherosclerosis. We chose to investigate statins because, despite being one of the first drugs designed with a specific molecular target in mind, statins have poorly understood pleiotropic effects. For example, in addition to lowering cholesterol by inhibiting HMG-CoA reductase, statins can reduce the risk of death from stroke. Statins can also have significant side effects including musculoskeletal deterioration and rhabdomyolysis, but how these deleterious effects occur is not known.
Statins are effective in inhibiting the yeast orthologs of HMG-CoA reductase and lowering levels of the yeast cholesterol analog, ergosterol. Statin treatment produces dose-dependent growth inhibition in yeast, presumably owing to the requirement for ergosterol for generation of new membrane. We screened our pilot metabolite library against a S. cerevisiae model of statin action. Our metabolite-statin screen revealed that the divalent metal ions zinc, copper and manganese were all effective in alleviating statin mediated growth inhibition. We characterized metal mediated statin rescue using an integrated approach that included biochemical, metabolomic and genomic approaches.
Metabolite profiling in yeast
Metabolism encompasses all the processes by which a cell generates energy and other essential molecules from nutrients. These pathways rely on hundreds of genes and involve thousands of small molecule intermediates, vitamins and cofactors. Interest in these molecules has led to development of technologies that allow high-throughput profiling of metabolic intermediates. We have optimized capillary electrophoresis methods for profiling amines, thiols and organic acids in the yeast Saccharomyces cerevisiae. Using these protocols, we have screened the yeast deletion collection and shown that clustering based on metabolite profiles allows us to identify related genes and pathways.
Figure 1: Amino acid profiling of a wild-type yeast extract using fluorescent derivatization of amine groups in combination with capillary electrophoresis separation.
Figure 2: Panel A shows amino acid profiling of the yeast deletion collection clustered by common metabolite profile. Panel B shows that arginine mutants show low levels of arginine and accumulation of arginine precursors such as ornithine and lysine. This cluster is also enriched for mitochondrial genes. Since arginine biosynthesis occurs in the mitochondria we propose that genes affecting mitochondrial function also affect arginine biosynthesis.
We have also begun metabolite profiling using gas chromatography and mass spectrometry (GCxGC-TOF). Preliminary experiments demonstrate that we can identify hundreds of unique compounds, including amino acids, sugars, organic acids and sterols. We are currently applying this method to better understand sterol biosynthesis in yeast. These complementary approaches provide a systematic view of metabolism in yeast. Other applications of this technology that we are working on include metabolite profiling in human urine samples from individuals with kidney disease and characterizing natural variation in yeast by assaying metabolic profiles along with transcription and protein levels in wild yeast strains.
Figure 3. Two-dimensional gas chromatography with mass spectrometry is used for identification and quantification of a couple of hundred intracellular small molecule metabolites.
•Sara Cooper and Sven Nelson (former lab members)
Cooper SJ, Finney GL, Brown SL, Nelson SK, Hesselberth J, Maccoss MJ, Fields S. High-throughput profiling of amino acids in strains of the Saccharomyces cerevisiae deletion collection. Genome Res. 2010 Sep;20(9):1288-96. download pdf
Functional chromosomal interactions
The topologies and spatial relationships of eukaryotic chromosomes are poorly understood. Together with the labs of Tony Blau, Bill Noble and Jay Shendure at the University of Washington, we developed a high-throughput method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of the budding yeast Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among tRNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.
Figure 1. Inter-chromosomal interactions. A, Circos diagram showing interactions between chromosome I and the remaining chromosomes. All 16 yeast chromosomes are aligned circumferentially, and arcs depict distinct inter-chromosomal interactions. Bold red hatch marks correspond to centromeres. B, Circos diagram, generated using the intra-chromosomal interactions depicting the distinct interactions between a small and a large chromosome (I and XIV, respectively). Most of the interactions between these two chromosomes primarily involve the entirety of chromosome I, and a distinct region of corresponding size on chromosome XIV.
Figure 2. Three-dimensional model of the yeast genome. Chromosomes are colored individually. Centromeres and telomeres are marked by lighter and darker red dots, respectively. All chromosomes cluster via centromeres at one pole of the nucleus (the area within the dashed oval), while chromosome XII extends outward toward the nucleolus, which is occupied by rDNA repeats (indicated by the white arrow). After exiting the nucleolus, the remainder of chromosome XII interacts with the long arm of chromosome IV.
•Kevin Schultz (former lab member)
Duan Z, Andronescu M, Schutz K, McIlwain S, Kim YJ, Lee C, Shendure J, Fields S, Blau CA, Noble WS. A three-dimensional model of the yeast genome. Nature. 2010 May 20;465(7296):363-7. download pdf
Capture and sequence analysis of RNAs containing 3' cyclic phosphate termini
Standard techniques used to isolate and identify RNA from cellular extracts have traditionally relied upon hybridization to oligo-dT or T4 RNA ligase-based methodologies. These methods have been successful in isolating populations of RNAs that are modified with poly-adenosine tracts or have hydroxyl moieties (-OH) at their 3’ terminus. It is possible that these two classes represent the majority of the cellular ‘RNA universe.’ However, with the development of advanced sequencing technologies, it is also clear that the RNA universe is more complex than previously appreciated. Therefore, there is a need to develop new technologies to further profile this complexity.
With this in mind we developed a technology that is capable of specifically isolating 2’,3’ cyclic phosphate-terminated RNAs from complex RNA mixtures. RNAs with these termini are generated as the product of particular RNA endonucleases or during ribonucleolytic cleavage. This technology uses the Arabidopsis thaliana tRNA ligase to add an adaptor oligonucleotide to RNAs that terminate in 2’,3’ cyclic phosphates. The adaptor allows specific priming by reverse transcriptase, which is followed by additional steps for PCR amplification and high throughput DNA sequencing. This method may identify processing events previously undetected by other RNA cloning techniques.
•Kevin Schutz (former lab member)
Schutz K, Hesselberth JR, Fields S. Capture and sequence analysis of RNAs with terminal 2',3'-cyclic phosphates. RNA. 2010 Mar;16(3):621-31. download pdf, supplemental figures, supplemental figure legends, supplemental raw seq data
Genomewide identification of transcription factor binding sites by DNAseI footprinting
The complement of DNA-binding proteins and their occupancy of sites throughout the genome determine an organism’s programs of gene expression, DNA replication and other chromosome-based processes. A detailed picture of factor binding on a genome-wide basis exists for Saccharomyces cerevisiae, obtained by a combination of transcriptional profiles, chromatin immunoprecipitation of more than 200 transcription factors, computational analyses and other assays. In an alternative approach, we have used digestion of chromatin by DNase I followed by high throughput DNA sequencing to identify sites of increased nuclease accessibility throughout the yeast genome. The resulting set of more than 10 million sequence reads provides both a global view of chromatin architecture as well as a gene-by-gene view of regulatory sequences protected from digestion by the presence of bound proteins. Unlike the case with results from chromatin immunoprecipitation, these gene-by-gene DNase I footprints can be used to directly identify transcription factor binding sites, and thereby infer their motifs. We found previously unknown binding sites in the genome for well-characterized factors, and observed other annotated binding sites that appear not to be protected from nuclease digestion under our conditions. This approach has the potential to characterize the transcriptional regulatory network of a poorly characterized organism given only its genome sequence.
Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, Fields S, Stamatoyannopoulos JA. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods. 2009 Apr;6(4):283-9. download pdf
•Jay Hesselberth & Zhihong Zhang (former lab members)
DrnI is a novel debranching enzyme-associated nuclease with a role in intron turnover
The turnover of introns spliced from pre-mRNA occurs first by debranching the lariat intron followed by destruction of the linear intron by other nucleases. We have identified a novel component of intron turnover, DRN1 (YGR093W). Using yeast two-hybrid screens, we found that Drn1 interacts with the debranching enzyme, Dbr1, and another spliceosomal component, Syf1. Sequence alignments revealed that Drn1 is homologous to the metallophosterase domain of Dbr1, and Drn1 has RNA endonuclease activity in vitro. Deletion of DRN1 results in the accumulation of lariat introns spliced from some ribosomal protein genes. We have identified genetic interactions between DRN1 and mutant alleles of PRP43, suggesting that Drn1 plays a role in the turnover of the spliceosomal complexes containing lariat introns. Intriguingly, the subset of Drn1-effected introns use RNA structural elements to stabilize conformations productive for splicing. We propose a model in which the nuclease activity of Drn1 is required for the efficient turnover of these large, structured introns, whose hyperstability may hinder the dissociation of lariat intron complexes by the spliceosomal DEAD/H-box ATPases.
•Jay Hesselberth & (former lab member)
Yeast Protein Interaction:
We constructed an array of ~6000 yeast transformants, each designed to express one of the S. cerevisiae open reading frames as a fusion to the Gal4 activation domain (AD). Using robotics, we can carry out a genomewide two-hybrid screen. A yeast strain expressing Gal4 DNA-binding domain (DBD) fused to any protein of interest is mated to the transformants in the array. Diploid cells are selected, pinned onto media selective for the two-hybrid interaction (-histidine) and scored for growth . Only cells that express interacting proteins should grow in the selective plates. The identity of interacting proteins is revealed by the positions of His+ colonies in the array. The initial description of the array, along with a collaborative effort by CuraGen, Inc. to carry out high throughput two-hybrid screens of yeast proteins, is described in Uetz et al. (2000).
*The data set can be downloaded here*
To date we have screened over 1000 yeast and several non-yeast proteins against the array. We are continuing this work on a collaborative basis through the Yeast Resource Center (YRC). Researchers with a specific gene of interest can contact us via the YRC website to request a collaboration. The gene of interest needs to be cloned into an appropriate two-hybrid DBD vector that we can provide. The identification of putative protein-protein interactions should further guide investigation of function.
In addition, we are working towards the development of a ‘dual-reporter’ Y2H system that should allow us to eliminate technical false positives that do not arise from a true interaction between the bait and prey proteins. Unlike the current 2-hybrid reporter system, this second reporter is not based on transcriptional activation, but rather on the reconstitution of a protein whose function can be selected or screened for. N-terminal and C-terminal fragments of proteins such as GFP and luciferase have been previously shown to function in split-reporter systems. We are in the process of fusing these domains to a well-established yeast two-hybrid protein pair and are currently testing different combinations in order to identify the optimal conditions that will allow us to distinguish between true and false positives.
Other publications describing yeast protein interactions that include work from the laboratory are listed here.
How the two-hybrid system works
How to clone 6000 ORFs
Primers • Vectors: pOBD2 and pOAD • Yeast strains • Recombination Cloning Protocols
•Sara Cooper and Sven Nelson (former lab members)
Dual-Reporter 2-Hybrid Assay:
Although the two-hybrid assay has been useful in discovering protein-protein interactions, a major disadvantage of the approach is the large number of false-positives that arise. Technical false positives activate the transcription of the HIS3 reporter gene but do not represent physical interaction of the two proteins (“bait” and “prey”). To address this issue, we have been working on incorporating a second reporter into the existing system. The second reporter relies on the re-association of the firefly luciferase protein, which has been split in two halves and fused to the bait and prey constructs. With a true bait and prey protein-protein interaction, the two halves of luciferase are brought into proximity and should reconstitute enzyme activity. Thus, true physical interactions should be both transcription positive and luminescence positive, whereas false positives would yield only the transcriptional signal.
Initial tests revealed that the two halves of luciferase were capable of self-associating even in the absence of interacting bait and prey proteins. Splitting of the luciferase protein also led to a large decrease in luminescence upon re-association of the two halves, compared to the intact protein. To address these issues, I performed random mutagenesis on the luciferase gene in order to identify mutations that could either boost luminescence or decrease self-association. Mutations from these two classes were then coupled and the resulting constructs are being tested on several known bait/prey protein pairs as well as on yeast prey proteins that are known false positives. Validation of this new system will require a two-hybrid screen of a library to be performed, yielding results that are enriched in true positives.
Random Addressed Protein Arrays
In the last decade, sequencing technologies have seen prodigious improvements in throughput and costs yet, protein activity and enzymatic assays and hence our ability to query gene function have not enjoyed concomitant advances. We are interested in applying the high-throughput sequencing technologies to generate an array of clusters containing DNA templates for in vitro transcription/translation (IVTT) reactions. Our goal is to identify each template in the array by sequencing and then generate a high-density protein array by capturing in vitro translated proteins proximal to the template cluster.
Towards this goal we have established methods for clonal amplification of DNA on solid surfaces (solid-phase amplification and emulsion amplification), in vitro transcription and translation methods using home-made cellular extracts, and are working on strategies for capturing proteins on surfaces.
Notably, an array platform containing millions of features will allow an increase in throughput three orders of magnitude above that of current protein arrays. Such increased throughput will enable experiments aimed at mapping protein interactions, generating enzyme activity profiles with activity-based probes and identifying novel enzyme activities to be performed at new scales. The ability to perform massively parallel protein assays should open novel research opportunities in proteomics, metagenomics and directed protein evolution. In addition, such development would also show how high-throughput sequencing platforms can be integrated into new technologies for functional assays, with end-points distinct from sequencing reads.
•Carlos Araya and Doug Fowler (former lab members)
Malaria Protein Arrays
Malaria is one of the world’s most devastating human diseases, affecting an estimated 500 million people and resulting in 2.5 million deaths globally each year. It is caused by four species of the protozoan parasite, Plasmodium, with the most deadly being Plasmodium falciparum. Progress in understanding malaria has been limited by the parasite’s complex life cycle and by difficulties expressing and purifying functional P. falciparum proteins in heterologous systems. With the completion of the P. falciparum genome sequence, a useful tool would be a protein array in which individual parasite proteins are displayed and available for functional assays.
To prepare a P. falciparum protein array, we are using 1000 P. falciparum open reading frames generated by the Structural Genomics of Pathogenic Protozoa (SGPP) consortium. These ORFs will be translated in vitro and printed onto glass slides. Our goal is to use these protein arrays in assays that benefit from rapid, simultaneous and sensitive screening of large numbers of proteins. For example, antigen profiling assays can identify P. falciparum proteins that govern host immune responses against the pathogen. Another application for the array is the biochemical characterization of the “hypothetical” proteins that represent more than 60% of P. falciparum genome, many of which are unique to P. falciparum. To characterize these proteins we will profile arrayed proteins with fluorescent substrate profiling probes.
•Anastasia Gridasova (former lab member)
Identifying protein targets of ubiquitin ligases
Ubiquitin (Ub) is a 76 amino acid protein that when attached to a target protein can alter its fate in multiple ways. Ub is an essential signaling molecule in nearly every pathway in eukaryotic cells. The E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases act in concert in order to covalently attach a Ub moiety to the epsilon amine on a lysine side chain or less commonly, the free amino group at the N-terminus of a substrate protein via an isopeptide bond. The E3 gives the enzyme cascade substrate specificity. There are 35 E3s in S. cerevisiae and nearly 1000 putative E3s in the human genome. Many proteins important for human disease are E3s, such as the breast cancer-specific tumor suppressor BRCA1 (breast cancer-1) and the early onset-Parkinson’s disease protein Parkin. Therefore, knowing the specific protein substrates of individual E3 enzymes would be of great importance in medicine.
One approach I am taking to determine specific substrates of E3s is as follows:
Individual E3 ubiquitin ligases will be fused to the bacterial biotin ligase, BirA, which attaches biotin to proteins that contain a biotin-acceptor peptide. A second fusion protein will be generated where ubiquitin is fused to a biotin-acceptor peptide fragment. In this arrangement, when the biotin-acceptor/ubiquitin fusion protein is brought to the E3 ubiquitin ligase/BirA fusion protein, the attached BirA will biotinylate the ubiquitin, thus marking it as having been acted on by that specific ubiquitin ligase. Substrate proteins to which the biotinylated ubiquitin is subsequently attached can then be purified using streptavidin chromatography and identified by mass spectrometry. Applying this approach to each individual E3 ubiquitin ligase will provide a critically needed proteome-wide view of ubiquitin ligases and their substrates. Ultimately, I would adapt the system for the study of ubiquitin ligases in mammalian tissue culture cells.
•Lea Starita & Russell Lo
Genomewide identification of spliced introns using a tiling microarray
One hallmark of eukaryotic gene structure is the presence of introns, which are spliced out of pre-mRNAs prior to translation. Introns excised from pre-mRNA molecules by the spliceosomal machinery are released in the form of lariats, in which the 5’ end of the intron RNA is linked via a phosphodiester bond to the 2’ hydroxyl of an internal adenosine residue. The lariat must be debranched by 2’-5’ phosphodiesterase prior to their turnover. In the absence or knockdown of the debranching enzyme, these lariat RNAs accumulate. We have carried out a genomewide identification of spliced intron using a genomic tiling microarray in Saccharomyces cerevisiaes by comparison of total RNA between DBR+ and dbr1 strains. This approach identified 141 of 272 known introns, confirmed three previously predicted introns, predicted four novel introns (of which two were experimentally confirmed), and led to the reannotation of four others.
DBR homologs and DBR-mediated lariat degradation were also found in other organisms. Currently, we are working on adapting the tiling array approach for genome-wide identification of introns in Drosophila and human cells. It has been reported that knockdown of the debranching enzyme in Drosophila via RNAi can cause lariat stabilization. We also applied this approach to human cell cultures and observed a similar but modest effect. We are now testing different RNAi knockdown approaches in both organisms to improve the efficiency of lariat accumulation. Analysis of lariat accumulation in these complex organisms will not only contribute to their genome annotation, but also extend our understanding of regulated and alternative splicing in these species.
•Zhihong Zhang & Jay Hesselberth (former lab members)
Zhang Z, Hesselberth JR, Fields S. Genome-wide identification of spliced introns using a tiling microarray.
Genome Res. 2007 Mar 9. download pdf, supplemental data
We are interested in turning the process of recombination to our ends, using it to facilitate gene therapy and genome engineering. To this end, we have developed a system that allows for the selection of yeast in which two overlapping parts of the selectable marker for Kanamycin (KanR) are brought together. Our system features a genomic recipient locus and a plasmid donor construct (Fig 1, new window). Recombination between these elements results in the reconstitution of a functional KanR marker when the elements are united through the mating of two yeast strains, each of which carries one of the two elements. We have made and tested a variety of donor structures (Fig 2,new window), together with different effector proteins that localize to the two DNA elements of the system.
The greatest total efficiency of conversion to the KanR phenotype was obtained with the donor construct having the most homology to the recipient locus. We are therefore using this construct to screen a random yeast genomic GAL4 library to isolate peptides that promote homologous repair. To do this, UAS GAL sequences have been cloned into the middle of the recipient locus and on the flanks of the longest donor construct we currently possess; yeast expressing peptides that promote HR when bound to the donor or recipient DNA will more frequently convert to the KanR phenotype. As genome engineering will most likely feature exogenously created linear donor DNA and may also utilize lesion-targeting endonucleases, we are employing the homing endonuclease I-SceI to introduce dsDNA breaks in both the donor and recipient DNA. While this will produce a background, we will screen serially derived libraries for clones enriched by repeated selection for the ability to promote HR.
•Clem Stanyon (former lab member)
The budding yeast Saccharomyces cerevisiae serves as a useful organism for studying factors that determine cellular longevity. The aging of mitotically active cells in higher eukaryotes can be modeled by the replicative life span of yeast mother cells, whereas aging of post-mitotic cells more closely resembles the chronological survival of quiescent yeast during stationary phase (Figure 1, new window). We are interested in using high-throughput technologies to identify and characterize genes that modify both aspects of cellular life span.
Measurement of yeast replicative life span requires micromanipulation of daughter cells away from mother cells following each mitotic cycle. The time-consuming nature of this assay has precluded large-scale analyses of replicative aging. In collaboration with Dr. Brian Kennedy (Department of Biochemistry, University of Washington), we have developed a method to allow semi-quantitative measurement of replicative life span based on the aging properties of a small number of cells. To date, we have determined the replicative life span phenotypes for approximately 20% (~1000 strains) of the ORF deletion collection. Completion of this analysis, in collaboration with the Kennedy lab, should take approximately 2 years.
Based on our analysis to date, we have already made several important discoveries, including the surprising finding that life span extension by calorie restriction (CR) does not require the NAD-dependent histone deacetylase, Sir2. The Sir2-indepenent nature of CR is demonstrated two ways: first, calorie restriction and overexpression of Sir2 increase life span additively, and second, CR increases life span to a greater extent in cells lacking Sir2 (and Fob1) than in wild type cells (Figure 2, new window).
Kaeberlein, M., Kirkland, K.T., Fields, S. and Kennedy, B.K. (2004) Sir2-independent life span extension by calorie restriction in yeast. PLoS Biology Sep;2(9):E296. download pdf
We have also determined that, contrary to a prior model proposed by Lin, Guarente, and colleagues, CR does not increase yeast life span by enhancing respiration. Yeast cells completely lacking mitochondrial DNA either have a normal life span or a dramatically shortened life span. In both cases, however, CR dramatically enhances longevity, demonstrating that respiration is not required for life span extension by CR.
Kaeberlein M, Hu D, Kerr EO, Tsuchiya M, Westman EA, Dang N, Fields S, Kennedy BK. Increased Life Span due to Calorie Restriction in Respiratory-Deficient Yeast. PLoS Genet. 2005 Nov 25;1(5):e69. download pdf
Of the first 564 single-gene deletion strains examined, 14 show a significant increase in replicative life span relative to the parental strain. This set includes two overlapping ORFs along with several genes that code for proteins with functions related to the nutrient responsive kinases Tor and Sch9. Of particular interest is the finding that three genes involved in ribosome biogenesis (a Tor and Sch9-regulated process) were among our set of long-lived deletion strains: REI1, RPL31A, and RPL6B. Rpl31a and Rpl6b are protein components of the large ribosomal subunit and Rei1 is a protein of unknown function that we have determined plays a role in large subunit biogenesis. This has led us to propose a model whereby CR increases life span by decreasing Tor and Sch9 activity which results in decreased ribosome biogenesis and translation (Figure 3, new window).
Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO, Kirkland KT, Fields S, Kennedy BK. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science. 2005 Nov 18;310(5751):1193-6. download pdf
In order to examine post-mitotic survival in a high-throughput manner, we have developed a method that allows for the simultaneous determination of chronological life span for several thousand yeast strains in a highly quantitative manner (Figure 4, new window). We have used this technology to screen the ORF deletion collection for genes whose deletion affects chronological aging. From this analysis, we have identified several genes (Table 1, new window) implicated in the TOR pathway that extend chronological life span when deleted (Figure 5, new window). The TOR proteins are highly conserved from yeast to humans and promote cellular growth in response to nutrients, especially amino acids. We have found that limitation of amino acids in the media, or pharmacological inhibition of TOR using rapamycin or methionine sulfoximine (MSX) (Figure 6, new window) can extend chronological life span, similar to deletion of TOR pathway components. Additionally, many of these interventions correlate with an increased nuclear accumulation of the stress-responsive transcription factor Msn2, and a resistance to heat and oxidative stresses. We propose a model by which decreased TOR activity up-regulates the activity of stress-response transcription factors (including Msn2) and thus promotes longevity (Figure 7, new window).
Powers RW 3rd, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 2006 Jan 15;20(2):174-84. download pdf
•Trey Powers and Matt Kaeberlein (former lab members)
Plasmodium Protein-Protein Interaction Project
Plasmodium falciparum is a mosquito-borne protozoan parasite responsible for the most severe form of malaria. Over 500 million people worldwide are afflicted with malaria, and each year more than one million people- most of them children - die from these infections. Despite the importance of malaria in global health, much remains to be discovered about the molecular biology of these pathogens. Of the ~5,300 proteins predicted from the P. falciparum genome sequence 60% are classified as hypothetical; this designation means that they have never been studied in Plasmodium and do not have sufficient similarity to characterized proteins in other organisms to allow functional assignments to be made. To begin to understand the functions of these novel proteins, we have identified a large number of protein-protein interactions using high-throughput yeast two-hybrid searches with protein fragments derived from genes expressed in the intraerythrocytic stages of P. falciparum. In collaboration with Prolexys Pharmaceuticals, we performed over 32,000 searches and we identified 2,846 interactions involving 1,308 proteins, which corresponds to approximately a quarter of the proteins predicted from the P. falciparum genome. We identified clusters of interacting proteins likely involved in important processes for the survival and infectivity of the parasite, such as gene regulation and host cell invasion. A large fraction of our interactions involve uncharacterized proteins and thus could lead to a new understanding of the functions of those proteins.
In addition to this, we have performed 10,000 searches with P. falciparum baits against human activation domain libraries, and 11,000 searches with P. vivax baits against P. vivax activation domain libraries. We are currently analyzing these datasets.
This work was funded in part by the Structural Genomics of Pathogenic Protozoans effort led by Wim Hol in the Department of Biochemistry.
•Doug LaCount and Marissa Vignali (former lab members)
Lacount DJ, Vignali M, Chettier R, Phansalkar A, Bell R, Hesselberth JR, Schoenfeld LW, Ota I, Sahasrabudhe S, Kurschner C, Fields S, Hughes RE. A protein interaction network of the malaria parasite Plasmodium falciparum. Nature. 2005 Nov 3;438(7064):103-7.
A network of WW domain interactions constructed using protein microarrays
We used protein microarray technology to generate a protein interaction map for twelve of the thirteen WW domains present in proteins of the yeast Saccharomyces cerevisiae (Example figure, new window). We observed a total of 1,158 interactions with these 12 domains, most of which have not previously been described. We analyzed the representation of functional annotations within the network, identifying enrichments for proteins with vacuolar and peroxisomal localization, as well as proteins involved in cofactor biosynthesis and protein turnover. The conservation of primary sequence motifs known to be recognized by WW domains was analyzed in the context of the network, and a comparative genomics approach used to dissect the occurrence of such motifs within the dataset. We analyzed the PY (Pro-Pro-Xaa-Tyr) motif in detail, and propose a novel consensus for the motif based on its conservation among orthologs of the interacting protein. The comparative approach revealed that one of the WW domain-containing proteins has an evolutionarily conserved PY motif, possibly indicating a role for WW domain multimerization in the propagation of signals derived from WW domain binding events.
•Jay Hesselberth and John Miller (former lab members)
Hesselberth JR, Miller JP, Golob A, Stajich JE, Michaud GA, Fields S. Comparative analysis of Saccharomyces cerevisiae WW domains and their interacting proteins. Genome Biol. 2006 Apr 10;7(4):R30. download pdf
Yeast Membrane Protein Array
We have applied the split-ubiquitin system originally described by Johnsson and Varshavsky (1994) to investigate interactions between integral membrane proteins. In brief, one protein is fused to the N-terminal half of ubiquitin (N-Ub) and a second protein is fused to the C-terminal half of ubiquitin (C-Ub). If the membrane proteins exist in close proximity, they bring the two halves of ubiquitin back together, and endogenous ubiquitin C-terminal hydrolases recognize this “reconstituted” ubiquitin and cleave the peptide bond following the last amino acid residue of ubiquitin. In the modified system of Stagljar et al (1998) there is a transcription factor fused to this residue and, upon cleavage, the transcription factor is released from the membrane to enter the nucleus to activate reporter genes.
We have generated a collection of 705 yeast proteins that are annotated as being in an “integral membrane” environment (643 proteins) along with proteins having amino acid homology to these (62). These proteins were made both as fusions with N-Ub, and as fusions to C-Ub with the transcription factor (C-UbPLV).
We tested our transformants for the successful insertion of an in-frame ORF into our C-UbPLV by screening them for an interaction with a generic “positive control”, a N-UbI fusion protein. Of the 705 proteins generated as C-UbPLV fusions, 365 showed an interaction with this wild-type version, N-UbI, which does not require a protein-protein interaction to bind to C-Ub. This result suggests that the 365 fusions bear an insert; the insert is in frame with the C-UbPLV moiety; there are no nonsense mutations in the insert; and the fusion protein is oriented such that the COOH-terminus bearing the fusion with C-UbPLV is exposed to the cytoplasm.
These 365 integral membrane proteins were screened for interactions against the full set of 705 proteins fused to N-UbG, a mutant form of the N-Ub with an isoleucine to glycine mutation at position 13 (Johnsson and Varshavsky (1994)). The pair-wise interactions between these 365 proteins and the 705 N-Ub fusion proteins were assayed similar to the two-hybrid study of Uetz et al. (2000). A set of 1985 putative interactions between 463 NubG fusions and 270 of the C-UbPLV fusions was found. This number of interactions per protein (~8 on average) is likely high due to the false-positive rate associated with this assay (the magnitude of which is not known). These false-positives are likely to result from the high effective concentration of integral membrane proteins due to both sequestration to a two-dimensional lipid bilayer, and co-transport of membrane proteins along the stages of the secretory pathway; additionally, over-expression of the fusion proteins from an episomal plasmid with an ectopic promoter will likely promote some non-physiological interactions.
An advantage of the membrane-based yeast two-hybrid system is that interactions between proteins can be detected at the physiological site of the interaction. This allows the observation of interactions occurring between proteins of most if not all subcellular membranous compartments (Figure1, new window). However, we also observe interactions between proteins whose native localization is to distinct compartments, and the system by itself is not informative in regards to the location within the cell of the interaction. In some instances (e.g., potentially the Mst27 and Tna1 interaction in the figure) the interaction may occur in early compartments of the secretory pathway, despite the mature proteins involved having disparate ultimate destinations. In other cases the observed interaction is likely to be non-physiological, and results from mislocalization of one or both of the proteins to an inappropriate compartment due to the fusion moiety.
In order to characterize our dataset with the goal of isolating those interactions that are more probably true-positives, we collaborated with Asa Ben-Hur in William Stafford Noble's group. The approach was to use a learning algorithm, the support vector machine (SVM), to classify the interactions based on the statistics of the assay as well as other datasets from the literature (e.g., synthetic-lethality, localization studies, Gene Ontology annotations, etc.). The SVM was trained using interactions found in this study that are also identified by independent experiments as examples of "true-positives". In addition to these 34 interactions, we included 22 that are supported by one computational approach (Deane et al., 2002), and 7 by a different computational analysis (Jansen et al., 2003). These 63 interactions constitute our highest confidence interactions, and are used by the SVM to identify features of the remaining interactions that support their classification as true interactions.
An interesting outcome from multiple SVM analyses is that 138 interactions are always classified as true positives by the algorithm, and 939 are never classified as true (Figure2, new window). We will therefore examine the interactions consistently predicted to be true as well as the intermediate interactions to identify potential physiological interactions. A comparison of the features of the 138 interactions that are always classified as true with those of the 138 "worst" interactions that are never classified as true shows that the SVM selects, for most features, the values that would be expected to indicate more physiological interactions as shown in the heat map (Figure3, new window).
•John Miller (former lab member) and Russell Lo
Miller J.P., Lo R.S., Ben-Hur A, Desmarais C, Stagljar I, Stafford Noble W, Fields S. Large-scale identification of yeast integral membrane protein interactions. Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12123-12128. supplemental data, download pdf
Specific Interests of the Yeast 2-Hybrid Array Screening
We were interested in using functional genomics tools to decipher two key biological processes that are dysregulated in cancer chromosome segregation and chromatin modifications. The focusing of genome-wide tools on specific biological processes has several benefits including: 1) bringing an unbiased approach to investigate the process; 2) discovering new players involved in a process; and 3) providing information to model the process. A further aspect of using focused functional genomics is we can come much closer to extracting information that is saturating for a particular method.
The core tool we used is the genome-wide two-hybrid array technology that has been successfully used in various projects in the lab and in collaboration with other labs. The projects that we have outlined below were of high interest.
Comprehensive two-hybrid interaction map of spindle-associated proteins:
The protein interaction information for kinetochore and associated spindle proteins is extensive but far from complete. Using the two-hybrid system, we started the comprehensive analysis of protein-protein interactions involving kinetochore and spindle proteins in collaboration with the Drubin and Barnes labs (UC Berkeley). These interactions are uncovering novel connections within the kinetochore and other cellular pathways.
Chromatin modification dependent protein interaction map:
Chromatin is subject to a variety of modifications and the specificity these modifications impart upon protein binding is still poorly understood. In collaboration with Min-Hao Kuo (Michigan State), we were working towards charting protein-protein interactions that are dependent on chromatin modifications. The combination of high-throughput techniques such as the tethered catalysis two-hybrid system and selective experiments will help elucidate the function of the various types of chromatin modifications.
Guo, D., Hazbun, T.R., Xu, X.-J., Ng, S.-L., Fields, S. and Kuo, M.-H. (2004) A tethered catalysis two-hybrid system to identify protein-protein interactions requiring post-translational modifications. Nature Biotechnology Jul;22(7):888-892. download pdf
Yeast Unknown ORF Project:
In a collaborative effort, the two-hybrid array technology was used in conjunction with groups affiliated with the YRC to decipher the roles of 100 essential and uncharacterized yeast genes (Hazbun et al., 2003). The integration of two-hybrid data with data from additional protein-based technologies such as co-purification and mass spectrometry, localization, and protein structure prediction enabled the functional annotation of a large fraction of these yeast genes. The parallel analysis of genes by four complementary technologies has also enhanced our understanding of the properties of the two-hybrid genome-wide array. The overlap of protein-protein interactions identified by mass spectrometry compared with two-hybrid was very low, although they both predicted similar cellular roles that agreed with localization data and protein structure prediction. The two-hybrid interactions tended to occur between proteins that were annotated in more broadly related biological processes, resulting in Go term assignments (Table 1, new window) that were lower in resolution than the mass spectrometry-based terms. This possibly reflects the tendency of two-hybrid to identify interactions with proteins in related biological processes that are not necessarily part of a core complex. For example, protein interactions identified by mass spectrometry for two unknown proteins suggested a role for inter-related complexes (Figure 1, new window) in DNA repair whereas two-hybrid interactions suggested a role in DNA repair as well as links with other biological processes such as chromosome segregation, sumoylation and ubiquitination.
Hazbun, T.R., Malmström, L., Anderson, S., Graczyk, B.J., Fox, B., Riffle, M., Sundin, B.A., Aranda, J.D., McDonald, W.H., Chun, C., Snydsman, B.E., Bradley, P., Muller, E.G.D., Fields, S., Baker, D., Yates, J.R. III and Davis, T.N. (2003) Assigning function to yeast proteins by integration of technologies. Molecular Cell 12:1353-1365. download pdf
•Tony Hazbun (former lab member)
A Yeast Screen for P. falciparum Mefloquine Resistance Genes
Mefloquine is an effective antimalarial drug. Unfortunately, resistant strains of Plasmodium falciparum are beginning to arise. The P. falciparum multi-drug resistant gene (Pfmdr1) encodes an ABC transporter that is often altered in mefloquine resistant strains and is presumed to act as a drug efflux pump. Little else is known about the parasite's mefloquine resistance mechanisms. However, mefloquine-resistant strains have been reported that contain no Pfmdr1 alterations, suggesting that additional genes are involved in mefloquine resistance. As the yeast Saccharomyces cerevisiae is sensitive to mefloquine, I have used it to screen for P. falciparum genes that can confer increased mefloquine resistance. Yeast was transformed with a P. falciparum cDNA library under the control of an S. cerevisiae galactose-inducible promoter, followed by selection on mefloquine. Several mefloquine resistance candidate genes were isolated in this screen. The four with the strongest phenotype were chosen for further analysis. These encode an uncharacterized multi-transmembrane-spanning protein, two small uncharacterized proteins, and a putative Rab GTPase activator. Each was analyzed for degree of mefloquine resistance and multidrug resistance. In addition, the mefloquine resistant P. falciparum strain W2-Mef and its sensitive parent W2 were analyzed by semi-quantitative RT-PCR to determine if any of these candidate genes is upregulated in the resistant strain. One candidate was thus regulated and it has been cloned for expression and drug testing in P. falciparum.
•Mara Jeffress (former lab member)
Jeffress M, Fields S. (2005) Identification of putative Plasmodium falciparum mefloquine resistance genes. Mol Biochem Parasitol. Feb;139(2):133-9. download pdf
Interactions of Human Toll-like Receptors
Toll-like receptor ‘sensor’ proteins are expressed in epithelia and antigen presenting cells. They are localized to the endoplasmic reticulum, the plasma membrane, and phagosome-lysosome membranes. The family of 10 human receptors, named TLR1 through TLR10, detects various microbial antigens or endogenous 'danger' signals, and subsequently triggers an ancient, highly conserved, innate immune response. TLRs are activated by ligand-induced oligomerization that recruits cytoplasmic signaling molecules to the receptors’ intracellular domains. Among the recruits are the MyD88 protein, and other adapters (TIRAP/Mal, TRIF/TICAM1, TRAM and SARM) that preferentially associate with certain activated receptors to impart some level of signaling specificity. All TLR cytoplasmic domains, as well as all identified signal adapters, harbor a canonical TIR (toll - interleukin - response) domain that mediates protein-protein interactions. All ten TLRs, as well as the tumor necrosis factor receptor and some interleukin receptors, activate the NFkB transcription factor. The proteins that impart specificity to the Toll signal transduction pathways are not fully delineated (Figure 1, new window).
Experiments in mammalian cells usually measure TLR activation by expression of an NFkB -driven reporter gene. However, any given cell may express many different receptors that use MyD88 as an adapter to activate NFkB, and of course other pathways, unrelated to the TLRs, can also activate NFkB. In contrast, the simple eukaryotic yeast S. cerevisiae does not possess endogenous TLRs or any recognizable Toll signaling pathway; therefore in yeast we can directly test protein-protein interactions without interference from endogenous proteins or other signaling pathways. We are expressing human TLR TIR domains in yeast, and using the yeast two-hybrid system to study protein interactions in the signal transduction pathway.
We screened for new proteins that bind to the receptors’ cytoplasmic domains, and have found many new interacting proteins that appear to specifically associate with certain TLR cytoplasmic domains. In particular, we have found novel and specific interactions for the closely related group of TLRs 1, 2, 6, & 10 (Table 1, new window). These are candidate proteins that may affect signaling by TLR2 heterocomplexes.
We also performed a structure-function study of the TLR-MyD88 interaction. We mapped the amino acids required for TLR association with this ‘universal’ adapter protein by swapping pieces of TLR2 into the closely related TLRs 1, 6, and 10. This creates chimaeric proteins with new (MyD88-binding) function, and is allowing us to define the exact amino acid differences between MyD88-binding and non-binding TLRs (Figure 2, new window).
The TLR-TIR domains are homologous to each other but not identical; therefore, examining protein interactions with these domains will lend insight into interaction specificity and how structure relates to function for the human TLRs.
•Victoria Brown-Kennerly and Rachel Brown (former lab members)
Brown V, Brown RA, Ozinsky A, Hesselberth JR, Fields S. Binding specificity of Toll-like receptor cytoplasmic domains. Eur J Immunol. 2006 Mar;36(3):742-53. download pdf
Genetic Screening Using Leucine Zippers
We developed a novel type of genetic screening method to identify proteins that function in a common pathway or process. The screen takes advantage of the observation that cellular processes are often initiated when a signal or upstream event causes two or more proteins to physically interact (Figure1, new window). These are usually part of a cascade of interactions that ultimately lead to the activation of the cellular process. We tested the idea that it might be possible to artificially force these interactions to occur and activate a process in the absence of its normal signal (i.e., cause a gain-of-function phenotype).
We were artificially forcing proteins to interact by fusing them to the leucine zippers from the mammalian Fos and Jun proteins (Figure2, new window). Fos and Jun leucine zippers form a stable heterodimer that can act as a tether to bring the attached proteins into close physical proximity. Using GFP and proteins that occur in specific subcellular localizations, we have shown that Fos and Jun can cause proteins to co-localize in yeast (Figure3, new window).
If artificial tethering mimics normal protein-protein interactions and recreates an activity that normally requires an upstream signal or event, it can then be used as a method to genetically screen for unknown members of a process (Figure4, new window). We can tether every yeast protein to a known component of the pathway (i.e. by coexpressing the known protein as a fusion with the Jun leucine zipper and a library of all yeast proteins fused to Fos) and look for a phenotype associated with activation of the process under study. The process should only be activated when the normal components are tethered to the known one. Since these will be fused to the Fos leucine zipper on a plasmid they can be easily identified.
A collection of all yeast proteins fused to the Fos leucine zipper might also be a useful reagent for tagging proteins. The tag can be added simply by introducing the tag (GFP, protein A, or any other polypeptide tag) fused to the Jun leucine zipper into yeast also expressing Fos fusions.
•Mike DeVit and Meg Branson (former lab members)
Devit M, Cullen PJ, Branson M, Sprague GF Jr, Fields S. (2005) Forcing interactions as a genetic screen to identify proteins that exert a defined activity. Genome Res. Apr;15(4):560-5. download pdf
Chemical Profiling of the Yeast Deletion Collection
Understanding the actions of drugs and toxins in a cell is of critical importance to medicine, yet many of the molecular events involved in chemical resistance are relatively uncharacterized. In order to identify the cellular processes and pathways targeted by chemicals, we took advantage of the haploid Saccharomyces cerevisiae deletion strains. Although ~4800 of the strains are viable, the loss of a gene in a pathway affected by a drug can lead to a synthetic lethal effect in which the combination of a deletion and a normally sublethal dose of a chemical results in loss of viability. We carried out genome-wide screens to determine quantitative sensitivities of the deletion set to four chemicals: hydrogen peroxide, menadione, ibuprofen, and mefloquine. Hydrogen peroxide and menadione induce oxidative stress in the cell, whereas ibuprofen and mefloquine are toxic to yeast by unknown mechanisms. Here we report the sensitivities of 659 deletion strains that are sensitive to one or more of these four compounds, including 163 multi-chemical sensitive strains, 394 strains specific to hydrogen peroxide and/or menadione, 47 specific to ibuprofen, and 55 specific to mefloquine. We correlate these results with data from other large-scale studies to yield novel insights into cellular function.
•Chandra Tucker (former lab member)
Tucker, C.L. and Fields, S. (2004) Quantitative genome-wide analysis of yeast deletion strain sensitivities to oxidative and chemical stress. Comparative and Functional Genomics 5:216-224. supplemental data, download pdf