Identification of protein‐protein interactions using <b><i>in vivo</i></b> cross‐linking and mass spectrometry

Vasilescu, Julian; Guo, Xuecui; Kast, Juergen

doi:10.1002/pmic.200400856

Cited by 205 publications

(195 citation statements)

References 36 publications

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“…Purified complexes can then be characterized by MS. Importantly and consistent with work by others (9,11,13), the reversed formaldehyde cross-links appear not to interfere with standard MS peptide identification.…”

supporting

confidence: 68%

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Tardiff

Abruzzi

Rosbash

2007

Proc. Natl. Acad. Sci. U.S.A.

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To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.mass spectrometry ͉ nascent RNP ͉ RNA processing ͉ transcription ͉ affinity purification P urification and mass spectrometric analysis of protein complexes is a ubiquitous approach in modern molecular biology (1). The identification of interacting proteins and their subsequent characterization provides valuable insight into the complexes that carry out essential cellular functions. Purifications can use multiple biochemical fractionations (e.g., ion exchange, gradients, gel filtration, and affinity purification) or a streamlined tandem affinity purification (TAP) approach (2). Conventional purification methods, however, often preclude the accurate identification of in vivo-relevant complexes. Major complications include dissociation of protein complexes, as a function of off-rates and extract dilution. Moreover, strong affinities can promote in vitro associations that do not exist in vivo. These can occur as a consequence of protein exchange and the absence of subcellular localization. Finally, major complexes may be insoluble, or harsh methods required to effect solubility can perturb complex composition. Ribonucleoprotein particles (RNPs) and especially transcription complexes are particularly subject to these caveats.The synthesis of protein-encoding mRNAs is directed by RNA polymerase II (pol II), an extensively studied macromolecular machine. The composition of pol II is dynamic and changes with the stage of transcription, namely, initiation, elongation, and termination (3, 4). Difficulties with complex purification underlie controversies, for example, to what extent initiation occurs by stepwise assembly of a preinitiation complex on DNA. Moreover, elongating pol II is tightly associated with DNA, which makes its characterization challenging (5).ChIP is a major tool for characterizing DNA-associated proteins and complexes, including transcription and cotranscriptional ...

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supporting

confidence: 68%

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Tardiff

Abruzzi

Rosbash

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Crosslinking was stopped with 0.1 M glycine (41). Cells were harvested in lysis buffer described above.…”

Section: Paraformaldehyde (Pfa) Crosslinkingmentioning

confidence: 99%

Characterization of the Physiological Turnover of Native and Inactivated Cytochromes P450 3A in Cultured Rat Hepatocytes: A Role for the Cytosolic AAA ATPase p97?

et al. 2007

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Mammalian hepatic cytochromes P450 (P450s) are endoplasmic reticulum (ER)-anchored hemoproteins engaged in the metabolism of numerous xeno-and endobiotics. P450s exhibit widely ranging half-lives, utilizing both autophagic-lysosomal (ALD) and ubiquitin-dependent 26S proteasomal (UPD) degradation pathways. Although suicidally inactivated hepatic CYPs 3A and "native" CYP3A4 in S. cerevisiae are degraded via UPD, the turnover of native hepatic CYPs 3A in their physiological milieu has not been elucidated. Herein, we characterize the degradation of native, dexamethasone-inducible CYPs 3A in cultured primary rat hepatocytes, using proteasomal and ALD [NH 4 Cl and 3-methyladenine (3-MA)] inhibitors to examine their specific degradation route. Pulse-chase cum immunoprecipitation analyses revealed a basal 52% 35 S-CYP3A loss over 6 h, which was stabilized by both proteasomal inhibitors. By contrast, no corresponding CYP3A stabilization was detected with either ALD inhibitor NH 4 Cl or 3-MA. Furthermore, MG-262-induced CYP3A stabilization was associated with its polyubiquitylation, thereby verifying that native CYPs 3A were also degraded via UPD. To identify the specific participants in this process, cellular proteins were crosslinked in situ with paraformaldehyde (PFA) in cultured hepatocytes. Immunoblotting analyses of CYP3A immunoprecipitates after PFA-crosslinking revealed the presence of p97, a cytosolic AAA ATPase instrumental in the extraction and delivery of ubiquitylated ER proteins for proteasomal degradation. Such native CYP3A-p97 interactions were greatly magnified after CYP3A suicidal inactivation (which accelerates UPD), and/or proteasomal inhibition, and were confirmed by proteomic and confocal immunofluorescence microscopic † Supported by NIH grants GM44037 (MAC) and DK26506 (MAC), RR012961 (KFM) and NIH grant P30DK26743 (Liver Core Center Cell and Tissue Biology Core). *Corresponding Author: M. A. Correia Dept. of Cellular and Molecular Pharmacology, Mission Bay Campus, Genentech Hall 600 16th Street, N572F/Box 2280 University of California San Francisco, CA 94158−2280 415−476−3992 (TEL) 415−476−5292 (FAX) e-mail: almira.correia@ucsf.edu. Supporting Information: MS/MS data obtained from the proteomic analyses of CYP3A-protein crosslinked complexes after subjection to SDS-PAGE, sequential gel-slicing of each relevant lane, and in situ tryptic digestion followed by LC-MS/MS analyses are provided. The proteins were identified from more than 1 peptide, but only 1 representative spectrum has been provided for each in the supplementary material. This material is available free of charge via the Internet at http://pubs.acs.org NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 15. Published in final edited form as:Biochemistry. analyses. These findings clearly reveal that native CYPs 3A undergo UPD and implicate a role for p97 in this process.The hepatic hemoproteins cytochromes P450 (P450s) 1 are key enzymes in the oxidative metabolism of various endob...

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“…Cell lysates were prepared in lysis buffer containing 25 mM lactose, 31 as described above, and the supernatants were subjected to immunoprecipitation. 42 Protein-binding assays. In GST pull-down experiments, cell lysates were incubated for 2 h at 41C with 2 mg purified GST or GST fusion proteins bound to glutathione beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK).…”

Section: Methodsmentioning

confidence: 99%

c-Abl and Arg tyrosine kinases regulate lysosomal degradation of the oncoprotein Galectin-3

Li¹,

Ma²,

Wang

et al. 2010

Cell Death Differ

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Galectin-3 (Gal3) has important roles in tumor transformation and metastasis. This study shows that c-Abl and Abl-related gene (Arg) associate with and phosphorylate Gal3. The SH (Src homology)3 domains of c-Abl/Arg bind to a P 80 GPPSGP motif of Gal3, and Tyr79 and Tyr118 are the major tyrosine phosphorylation sites. A consequence of this interaction and phosphorylation is the significant impairment of chaperone-mediated autophagy of Gal3. Cells expressing Gal3 and treated with the c-Abl/Arg inhibitor STI571, Gal3-depleted cells, and Gal3-depleted cells expressing Gal3 phosphorylation mutants all display an increased sensitivity to apoptosis-inducing agents. In addition, tumor cells expressing the phosphorylation mutants show impaired tumorigenicity. These results partially explain the antiapoptotic effect of Abl and Arg. As tumors frequently overexpress Gal3, a c-Abl/Arg-specific inhibitor may potentially be applied along with other antitumor drugs to target the lysosomal degradation of Gal3 in tumor therapy.

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Identification of protein‐protein interactions using in vivo cross‐linking and mass spectrometry

Cited by 205 publications

References 36 publications

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking

Characterization of the Physiological Turnover of Native and Inactivated Cytochromes P450 3A in Cultured Rat Hepatocytes: A Role for the Cytosolic AAA ATPase p97?

c-Abl and Arg tyrosine kinases regulate lysosomal degradation of the oncoprotein Galectin-3

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