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Showing papers by "Steven P. Gygi published in 2001"


Journal ArticleDOI
TL;DR: It is shown thatosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF3E, and a novel combination of two-dimensional isoelectric focusing and Western blotting with phosphospecific antibodies is established.
Abstract: In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5′ end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.

851 citations


Journal ArticleDOI
TL;DR: Current approaches to identify and characterize large numbers of proteins and measure dynamic changes in protein expression directly from complex protein mixtures (total cell lysates) are addressed.
Abstract: Proteomics can be defined as the systematic analysis of proteins for their identity, quantity and function. In contrast to a cell's static genome, the proteome is both complex and dynamic. Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). However, this technique is under scrutiny because of a failure to detect low-abundance proteins from the analysis of whole cell lysates. Alternative approaches integrate a diversity of separation technologies and make use of the tremendous peptide separation and sequencing power provided by MS/MS. When liquid chromatography is combined with tandem mass spectrometry (LC/MS/MS) and applied to the direct analysis of mixtures, many of the limitations of 2DE for proteome analysis can be overcome. This tutorial addresses current approaches to identify and characterize large numbers of proteins and measure dynamic changes in protein expression directly from complex protein mixtures (total cell lysates).

631 citations


Journal ArticleDOI
TL;DR: In this paper, the authors have identified and characterized an alternative RFC complex RFC(CTF18p, CTF8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission.

298 citations


Journal ArticleDOI
TL;DR: The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.
Abstract: We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag reagents (Gygi, S. P.; et al. Nat. Biotechnol. 1999, 17, 994-9.). Proteins in mixtures are first labeled on cysteinyl residues using an isotope coded affinity tag reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multidimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reversed-phase liquid chromatography column directly onto a MALDI sample target. After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification. The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.

181 citations


Journal ArticleDOI
TL;DR: The results suggest that Tb MP52 and TbMP48 are components of the RNA editing complex.
Abstract: RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3*-terminal uridylyl transferase, 3*-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an ;49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.

120 citations


Journal ArticleDOI
TL;DR: Results indicate that these four proteins are components of the RNA editing complex and that Tb MP63 and TbMP52 can interact.
Abstract: RNA editing in kinetoplastid mitochondria occurs by a series of enzymatic steps that is catalyzed by a macromolecular complex. Four novel proteins and their corresponding genes were identified by mass spectrometric analysis of purified editing complexes from Trypanosoma brucei. These four proteins, TbMP81, TbMP63, TbMP42, and TbMP18, contain conserved sequences to various degrees. All four proteins have sequence similarity in the C terminus; TbMP18 has considerable sequence similarity to the C-terminal region of TbMP42, and TbMP81, TbMP63, and TbMP42 contain zinc finger motif(s). Monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities. The proteins are present in the immunoprecipitates and sediment at 20S along with the in vitro editing, and RNA editing ligases TbMP52 and TbMP48. Recombinant TbMP63 and TbMP52 coimmunoprecipitate. These results indicate that these four proteins are components of the RNA editing complex and that TbMP63 and TbMP52 can interact. RNA editing in trypanosomes posttranscriptionally inserts and deletes uridylates (U’s) at multiple sites in most mitochondrial pre-mRNAs to produce mature mRNAs. U insertion and deletion are directed by guide RNAs (gRNAs) and are catalyzed by a macromolecular complex. Editing occurs by a series of enzymatic steps that include endoribonuclease, 3 terminal uridylyl transferase (TUTase), 3 exouridylylase, and RNA ligase activities (reviewed in references 3, 8, 21, and 23). Although editing can be extensive, with the insertion and deletion of numerous U’s, it is also very specific. The characteristics of the enzymatic activities contribute to this specificity (7), but noncatalytic proteins may be required for editing and may contribute to the specificity. RNA editing is catalyzed by a 20S ribonucleoprotein complex (2, 15), and identification of its components and the composition of the fully functional complex is at an early stage. Initial studies estimated that a complex that can catalyze at least some of the steps of editing in vitro contains 7 to 20 polypeptides (11, 14, 16). Two related proteins, TbMP52 and TbMP48, were identified by mass spectrometric analysis of purified editing complexes (14), and TbMP52 was shown to be essential for RNA editing and for survival of bloodstream forms in vivo and in vitro (19). In addition, TbMP52 and TbMP48 correspond to the larger and smaller adenylatable proteins in the RNA editing complex, respectively, and were found to be RNA ligases (12, 17, 19). TbMP52 corresponds to T. brucei V and T. brucei p52 and TbMP48 corresponds to T. brucei IV and T. brucei p48 (12, 17). The fully functional editing complex, which may consist of a catalytic core complex and accessory and regulatory factors may contain numerous proteins. Several other candidate proteins, some of which have RNA binding activities, have been described and may play a role in RNA editing (6, 9, 11, 13, 25). However, except for mHel61p (13), none of these proteins have been shown to play a direct role in editing. In this study, we describe the identification of four additional proteins that are present in the RNA editing complex by immunoprecipitation, mass spectrometric, and/or gradient sedimentation analyses. These four proteins have sequence similarities to each other and the three largest contain one or two C2H2 zinc finger motif(s). One protein was also shown to interact in vitro with the TbMP52 editing RNA ligase.

113 citations


Journal ArticleDOI
TL;DR: A new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance.

94 citations


Journal ArticleDOI
TL;DR: It is shown here that proximity of the IUR element to the adjacent NF-κB element is critical to its function as a positive regulatory element and suggests that PARP may be a coactivator of CXCL1 transcription.

89 citations