8 research outputs found
Ras Interaction with Two Distinct Binding Domains in Raf-1 5 Be Required for Ras Transformation
Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131)
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A Quest for Purpose: Investigating the Functional Implication of the Methylation of Proteins Involved in Translation
Methylation is an essential post translational modification that can be found on a variety of proteins in higher and lower species. The effects of these modifications are diverse and include targeting genes for activation or silencing and protein stability. The most extensively characterized methylation reaction is that of histone tails. In the past decade researchers became interested in identifying non-histone protein substrates and the enzymes responsible for methylating them (methyltransferases). In the yeast Saccharomyces cerevisiae, a substantial number of methyltransferases have been identified for modifying mitochondrial proteins, translational release factors, ribosomal proteins and translational elongation factors. When I started my doctoral journey the newest methyltransferase of the yeast translational apparatus was found to modify elongation factor 1 alpha (EF1A), the protein responsible for bringing the aminoacyl-tRNA to the ribosome. This enzyme, Efm7, was the fifth elongation factor methyltransferase (Efm) responsible for methylating EF1A! EF1A methylation by five methyltransferases is unique as other elongation factors, EF2 and EF3, only have two methyltransferases that modify them both. Additionally, each of these five EF1A methyltransferases appears to target specific residues on EF1A. This has not been seen in any other protein of the translational apparatus that is methylated. Thus I was captivated by this uniqueness of EF1A methylation.Although it is known that EF1A and other translational proteins are methylated and the enzymes responsible for its methylation, we still don’t know why it is methylated. GTP-bound EF1A functions as the courier of aminoacylated tRNA to the ribosome. When a correct codon-anticodon match is made, a conformational change in the ribosome results in the hydrolysis of GTP to GDP and the release of EF1A. Additionally, GDP bound-EF1A functions outside of the ribosome including being able to bind and bundle filamentous actin. This dissertation focused on identifying whether or not there was a direct link between methylation and functionality of EF1A. In a separate study, I examined the functional role of ribosomal protein methyltransferases.My studies lead to the development and characterization of the first quintuplet knockout yeast strain for the enzymes responsible for methylating EF1A. These strains have been confirmed to be methylation deficient at the respective EF1A lysine sites, using mass spectrometry. I found that EF1A methylation appears to be necessary for helping yeast to survive and adapt to differences in its cellular environment but may not be necessary for the integrity of the actin cytoskeleton as well as EF1A induced actin binding
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Protein Methylation and Translation: Role of Lysine Modification on the Function of Yeast Elongation Factor 1A.
To date, 12 protein lysine methyltransferases that modify translational elongation factors and ribosomal proteins (Efm1-7 and Rkm 1-5) have been identified in the yeast Saccharomyces cerevisiae. Of these 12, five (Efm1 and Efm4-7) appear to be specific to elongation factor 1A (EF1A), the protein responsible for bringing aminoacyl-tRNAs to the ribosome. In S. cerevisiae, the functional implications of lysine methylation in translation are mostly unknown. In this work, we assessed the physiological impact of disrupting EF1A methylation in a strain where four of the most conserved methylated lysine sites are mutated to arginine residues and in strains lacking either four or five of the Efm lysine methyltransferases specific to EF1A. We found that loss of EF1A methylation was not lethal but resulted in reduced growth rates, particularly under caffeine and rapamycin stress conditions, suggesting EF1A interacts with the TORC1 pathway, as well as altered sensitivities to ribosomal inhibitors. We also detected reduced cellular levels of the EF1A protein, which surprisingly was not reflected in its stability in vivo. We present evidence that these Efm methyltransferases appear to be largely devoted to the modification of EF1A, finding no evidence of the methylation of other substrates in the yeast cell. This work starts to illuminate why one protein can need five different methyltransferases for its functions and highlights the resilience of yeast to alterations in their posttranslational modifications
Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae : Roles in ribosome biogenesis and translation
A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed −1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation
Hubble Space Telescope Spectroscopic Observations of the Narrow-Line Region in Nearby Low-Luminosity Active Galactic Nuclei
Original article can be found at: http://www.iop.org/EJ/journal/aj Copyright American Astronomical Society DOI: 10.1088/0004-6256/136/4/1677 [Full text of this article is not available in the UHRA]We present Space Telescope Imaging Spectrograph observations of 14 nearby low-luminosity active galactic nuclei, including 13 LINERs and 1 Seyfert, taken at multiple parallel slit positions centered on the galaxy nuclei and covering the Hα spectral region. For each galaxy, we measure the emission-line velocities, line widths, and strengths to map out the inner narrow-line region structure—typically within ~100 pc from the galaxy nucleus. There is a wide diversity among the velocity fields: in a few galaxies the gas is clearly in disk-like rotation, while in other galaxies the gas kinematics either appear chaotic or are dominated by radial flows with multiple velocity components. In most objects, the emission-line surface brightness distribution is very centrally peaked. The [S II] line ratio indicates a radial stratification in gas density, with a sharp increase within the inner 10-20 pc, in the majority of the Type 1 (broad-lined) objects. The electron-density gradients of the Type 1 objects exhibit a similar shape that is well fit by a power law of the form n e = n 0(r/1 pc)α, where α = –0.60 ± 0.13. We examine how the [N II] λ6583 line width varies as a function of the aperture size over a range of spatial scales, extending from scales comparable to the black hole's sphere of influence to scales dominated by the host galaxy's bulge. For most galaxies in the sample, we find that the emission-line velocity dispersion is largest within the black hole's gravitational sphere of influence, and decreases with increasing aperture size toward values similar to the bulge stellar velocity dispersion measured within ground-based apertures. We construct models of gas disks in circular rotation and show that this behavior can be consistent with virial motion, although for some combinations of disk parameters we show that the line width can increase as a function of aperture size, as observed in NGC 3245. Future dynamical modeling to determine black hole masses for a few objects in this sample may be worthwhile, although disorganized motion will limit the accuracy of the mass measurements.Peer reviewe