Biochemical and genetic analysis of the yeast proteome with a movable ORF collection

Gelperin, Daniel; White, Michael A.; Wilkinson, Martha L.; Kon, Yoshiko; Kung, Li; Wise, Kevin J.; Lopez-Hoyo, Nelson; Jiang, Lixia; Piccirillo, Stacy; Yu, Haiyuan; Gerstein, Mark; Dumont, Mark E.; Phizicky, Eric M.; Snyder, M; Grayhack, Elizabeth J.

doi:10.1101/gad.1362105

Cited by 458 publications

(523 citation statements)

References 60 publications

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“…Labeled tRNA Gly was incubated at 30°C for 18 h in 10 mL reaction mixtures containing S-adenosylmethionine and 2 mL (z0.7 mg) protein from 59 pools of purified fusion proteins from the MORF collection (numbered 1-53, 55, 56, 58, 59, 64, and 66), as indicated, and analyzed as described in B and Materials and Methods. reading frame (MORF) library of yeast strains, each of which expresses a yeast ORF fused at its C terminus to a tri-partite affinity tag (Gelperin et al 2005). To do this, we used the biochemical genomics approach described previously for another genomic library (Xing et al 2002;Gu et al 2003;Jackman et al 2003;Alexandrov et al 2005), employing pools of purified proteins.…”

Section: Resultsmentioning

confidence: 99%

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Wilkinson¹,

Crary²,

Jackman³

et al. 2007

RNA

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The methylation of the ribose 29-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 29-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 29-O-methylation that occurs in the middle of a duplex region in tRNA, the modification is conserved in eukaryotes, and the features of the tRNA necessary for substrate recognition are poorly defined. We show here that Saccharomyces cerevisiae ORF YOL125w (TRM13) is necessary and sufficient for 29-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 29-O-methylation activity, using tRNA Gly(GCC) as a substrate, and extracts made from a trm13-D strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-D strain lack the corresponding 29-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 29-O-methylation at position 4 in vitro since yeast Trm13 protein purified after expression in Escherichia coli has the same activity as that produced in yeast. Trm13 protein binds substrates tRNA His and tRNA Gly(GCC) with K D values of 85 6 8 and 100 6 14 nM, respectively, and has a K M for tRNA His of 10 nM, but binds nonsubstrate tRNAs very poorly (K D > 1 mM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.

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Section: Resultsmentioning

confidence: 99%

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Wilkinson¹,

Crary²,

Jackman³

et al. 2007

RNA

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“…Samples were then mixed with 23 Laemmli sample buffer (Bio-Rad) and heated at 95°f or 5 min, following which 18 ml was loaded on either an 8-16% or a 10% polyacrylamide gel (Bio-Rad). SDS-PAGE was carried out at 150 V. Proteins were transferred to a PVDF membrane and detected with a rat monoclonal anti-HA primary antibody (clone 3F10, Roche) and a goat anti-rat HRP-conjugated secondary antibody ( Jackson ImmunoResearch), as previously described (Gelperin et al 2005). The blot was developed with the ECL Advance kit (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

“…The wild-type strains used as controls in the flow cytometry experiments were the deletion collection parental strains: BY4743 (MATa/a, his3D1/his3D1, leu2D0/leu2D0, MET15/met15D0, LYS2/ lys2D0, ura3D0/ura3D0), BY4741 (MATa,his3D1,leu2D0,met15D0,ura3D0), and BY4742 (MATa,his3D1,leu2D0,lys2D0,ura3D). The P GAL1 -SFG1 overexpression strain used in the expression profiling experiments was taken from the MORF yeast ORF collection (Gelperin et al 2005), available at Open Biosystems; the host strain for this collection is Y258 (MATa,112) (Zhu et al 2001).…”

Section: Methodsmentioning

confidence: 99%

A Systematic Screen for Transcriptional Regulators of the Yeast Cell Cycle

2009

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Transcription factors play a key role in the regulation of cell cycle progression, yet many of the specific regulatory interactions that control cell cycle transcription are still unknown. To systematically identify new yeast cell cycle transcription factors, we used a quantitative flow cytometry assay to screen 268 transcription factor deletion strains for defects in cell cycle progression. Our results reveal that 20% of nonessential transcription factors have an impact on cell cycle progression, including several recently identified cyclin-dependent kinase (Cdk) targets, which have not previously been linked to cell cycle transcription. This expanded catalog of cell-cycle-associated transcription factors will be a valuable resource for decoding the transcriptional regulatory interactions that govern progression through the cell cycle. We conducted follow-up studies on Sfg1, a transcription factor with no previously known role in cell cycle progression. Deletion of Sfg1 retards cells in G 1 , and overexpression of Sfg1 delays cells in the G 2 /M phase. We find that Sfg1 represses early G 1 , Swi5/Ace2-regulated genes involved in mother-daughter cell separation. We also show that Sfg1, a known in vitro cyclin-dependent kinase target, is phosphorylated in vivo on conserved Cdk phosphorylation sites and that phosphorylation of Sfg1 is necessary for its role in promoting cell cycle progression. Overall, our work increases the number of transcription factors associated with cell cycle progression, strongly indicates that there are many more unexplored connections between the Cdk-cyclin oscillator and cell cycle transcription, and suggests a new mechanism for the regulation of cell separation during the M/G 1 phase transition.

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“…With regard to the latter, the yeast genome contains approximately 1130 essential genes, and we readily envision application of our platform to this important yeast gene set. The resulting mutant collection will provide a strong complement to existing genome-wide collections of titratable promoter alleles (Mnaimneh et al, 2004), gene deletion mutants (Winzeler and Davis, 1997;Winzeler et al, 1999) and gene overexpression strains (Sopko et al, 2006;Gelperin et al, 2005). Similarly, genome-scale mislocalization screens will augment pathway discovery tools, such as synthetic genetic array analysis (Tong et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

A small molecule‐directed approach to control protein localization and function

Geda

Patury

et al. 2008

Yeast

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Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus. The kinetics of this system are rapid: rapamycin-directed nucleo-cytoplasmic transport is evident 10-12 min posttreatment and the process is reversible upon removal of rapamycin. Accordingly, we envision this platform as a promising approach for the systematic construction of conditional loss-of-function mutants. As proof of principle, we used this system to direct nuclear export of the essential heat shock transcription factor Hsf1p, thereby mimicking the cell-cycle arrest phenotype of an hsf1 temperature-sensitive mutant. Our drug-induced localization platform also provides a method by which protein localization can be uncoupled from endogenous cell signalling events, addressing the necessity or sufficiency of a given localization shift for a particular cell process. To illustrate, we directed the nuclear import of the calcineurin-dependent transcription factor Crz1p in the absence of native stimuli; this analysis directly substantiates that nuclear translocation of this protein is insufficient for its transcriptional activity. In total, this technology represents a powerful method for the generation of conditional alleles and directed mislocalization studies in yeast, with potential applicability on a genome-wide scale.

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Biochemical and genetic analysis of the yeast proteome with a movable ORF collection

Abstract: [Keywords: Biochemical genomics; protein microarray; proteome; high-throughput expression; galactose lethality; glycosylation] Supplemental material is available at http://www.genesdev.org.

Cited by 458 publications

References 60 publications

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

A Systematic Screen for Transcriptional Regulators of the Yeast Cell Cycle

A small molecule‐directed approach to control protein localization and function

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