Tandem affinity purification and identification of protein complex components

Gould, Kathy

doi:10.1016/s1046-2023(03)00318-9

Cited by 22 publications

(23 citation statements)

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“…The general procedure for TAP has been discussed elsewhere (Puig et al, 2001; Cox et al, 2002; Bouwmeester et al, 2004; Gould et al, 2004). We have followed the established protocols with only minor modifications.…”

Section: Methods and Resultsmentioning

confidence: 99%

Analyzing protein complexes in Drosophila with tandem affinity purification–mass spectrometry

Veraksa

Bauer²,

Artavanis‐Tsakonas

2005

Developmental Dynamics

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We describe the application of tandem affinity purification-mass spectrometry (TAP-MS) to the study of protein complexes in Drosophila. We have constructed vectors for inducible expression of TAP-tagged fusion proteins in Drosophila cultured cells and in vivo. Using these vectors, we tagged, as a paradigm, several components of the Notch signaling pathway, isolated protein complexes containing the baits and associated proteins from cells and embryos, and identified the subunits by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several known interactions involving Notch pathway elements were confirmed, and many novel potential interactions were uncovered. For some of the novel associations, we validated the interaction genetically and biochemically. We conclude that TAP, in combination with MS, can be used as an effective method for the studies of the Drosophila proteome. Developmental Dynamics 232: 827-834, 2005.

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Section: Methods and Resultsmentioning

confidence: 99%

Analyzing protein complexes in Drosophila with tandem affinity purification–mass spectrometry

Veraksa

Bauer²,

Artavanis‐Tsakonas

2005

Developmental Dynamics

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“…The E1‐activating enzyme and E2 (Ubc13/Uev1a) were purchased from Boston Biochem. Dma1–HA 3 –TAP and Dma1(I194A)–HA 3 –TAP were purified from S. pombe lysates using a tandem affinity purification method (Gould et al , 2004). All components were incubated in a reaction buffer containing 50 mM Tris–HCl (pH 7.5), 2.5 mM MgCl 2 and 0.5 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

Dma1 ubiquitinates the SIN scaffold, Sid4, to impede the mitotic localization of Plo1 kinase

Johnson

Gould

2010

The EMBO Journal

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Proper cell division requires strict coordination between mitotic exit and cytokinesis. In the event of a mitotic error, cytokinesis must be inhibited to ensure equal partitioning of genetic material. In the fission yeast, Schizosaccharomyces pombe, the checkpoint protein and E3 ubiquitin ligase, Dma1, delays cytokinesis by inhibiting the septation initiation network (SIN) when chromosomes are not attached to the mitotic spindle. To elucidate the mechanism by which Dma1 inhibits the SIN, we screened all SIN components as potential Dma1 substrates and found that the SIN scaffold protein, Sid4, is ubiquitinated in vivo in a Dma1-dependent manner. To investigate the role of Sid4 ubiquitination in checkpoint function, a ubiquitination deficient sid4 allele was generated and our data indicate that Sid4 ubiquitination by Dma1 is required to prevent cytokinesis during a mitotic checkpoint arrest. Furthermore, Sid4 ubiquitination delays recruitment of the Polo-like kinase and SIN activator, Plo1, to spindle pole bodies (SPBs), while at the same time prolonging residence of the SIN inhibitor, Byr4, providing a mechanistic link between Dma1 activity and cytokinesis inhibition.

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“…The extraction buffer was IPP-buffer (10 mM Tris-Cl, pH.7.9, 150 mM NaCl, 0.1% Nonidet (NP-40), 0.5 mM DTT, 0.5 mM phenylmethyl sulphonide fluoride, 10 lg/ml leupeptin, 10 lg/ml antipain). The cells were lysed in 20 ml of IPPbuffer using glass beads (0.5 mm diameter size) in Fast Prep apparatus (Biospec from Q-Biogen) according to Gould et al (2004). After the extraction, the suspension was diluted three times with the IPP-buffer, centrifuged at 3,000g for 5 min, followed by a second centrifugation of the supernatant at 27,000g.…”

Section: Tap-knr4 Gene Fusion and Purification Of Tap-tagged Knr4mentioning

confidence: 99%

The ‘interactome’ of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae

et al. 2005

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The integrity of the Saccharomyces cerevisiae cell wall requires a functional Pkc1-Slt2 MAP kinase pathway that contributes to transient growth arrest, enabling coordination of cell division with cell wall remodelling. How this coordination takes place is still an open question. Recently, we brought evidence that Knr4 protein, whose absence leads to several cell wall defects, may play a role in this function. Here, we show that Knr4 is a monomeric protein that exhibits an aberrant mobility on a SDS-gel electrophoresis and a non-globular structure. Furthermore, Knr4 is an unstable protein that is degraded as cells enter the stationary phase of growth, while its corresponding gene is constitutively expressed. In exponentially growing cells on glucose, Knr4 appeared to be present in a protein complex that migrates with an apparent Mw superior to 250 kDa. Using the TAP-tag methodology, nine potential partners of Knr4 were identified, which could be distributed into three biological processes. A first group consisted of Slt2 and Pil1, two proteins dedicated to cell wall maintenance and biogenesis. The second group comprised four proteins (Bud6, Act1, Cin8 and Jnm1) implicated in the establishment of cell polarity and bud integrity during mitosis. The last group contained four proteins (Asc1, Ubc1, Hsc82 and Gvp36) that probably deal with the stability/degradation of proteins. Deletion analysis revealed that the domain of interaction covered 2/3 of the Knr4 sequence on the N-terminal side. Moreover, the replacement of the two in vivo phosphorylated Ser(200) and Ser(203) by alanines led to a mutated protein with reduced protein interactions and a weaker complementation ability towards knr4 null mutant phenotypes. These results together with previous data from genome scale two-hybrid and synthetic interaction screens support the notion that Knr4 is a regulatory protein that participates in the coordination of cell wall synthesis with bud emergence, and that this function may be modulated by phosphorylation of this protein.

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Tandem affinity purification and identification of protein complex components

Cited by 22 publications

References 0 publications

Analyzing protein complexes in Drosophila with tandem affinity purification–mass spectrometry

Analyzing protein complexes in Drosophila with tandem affinity purification–mass spectrometry

Dma1 ubiquitinates the SIN scaffold, Sid4, to impede the mitotic localization of Plo1 kinase

The ‘interactome’ of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae

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