Sequential Peptide Affinity (SPA) System for the Identification of Mammalian and Bacterial Protein Complexes

Zeghouf, Mahel; Li, Joyce; Butland, Gareth; Borkowska, Anna; Canadien, Veronica; Richards, Dawn P.; Beattie, Bryan K.; Emili, Andrew; Greenblatt, Jack

doi:10.1021/pr034084x

Cited by 191 publications

(198 citation statements)

References 18 publications

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“…To this end, sequential peptide affinity (SPA)-tagged DprA [i.e., harboring a C-terminal Flag epitope separated from a calmodulin-binding domain by a cleavage site for the TEV protease (19)] was ectopically expressed under the control of a competence-inducible promoter (Materials and Methods). The resulting ectopic construct was found to complement fully the transformation defect of a dprA-null mutant strain, demonstrating that the DprA-SPA fusion is functional.…”

Section: Resultsmentioning

confidence: 99%

Structure–function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation

Quevillon‐Chéruel

Campo

Mirouze

et al. 2012

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

154

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Transformation promotes genome plasticity in bacteria via RecAdriven homologous recombination. In the Gram-positive human pathogen Streptococcus pneumoniae, the transformasome a multiprotein complex, internalizes, protects, and processes transforming DNA to generate chromosomal recombinants. Double-stranded DNA is internalized as single strands, onto which the transformation-dedicated DNA processing protein A (DprA) ensures the loading of RecA to form presynaptic filaments. We report that the structure of DprA consists of the association of a sterile alpha motif domain and a Rossmann fold and that DprA forms tail-totail dimers. The isolation of DprA self-interaction mutants revealed that dimerization is crucial for the formation of nucleocomplexes in vitro and for genetic transformation. Residues important for DprARecA interaction also were identified and mutated, establishing this interaction as equally important for transformation. Positioning of key interaction residues on the DprA structure revealed an overlap of DprA-DprA and DprA-RecA interaction surfaces. We propose a model in which RecA interaction promotes rearrangement or disruption of the DprA dimer, enabling the subsequent nucleation of RecA and its polymerization onto ssDNA.bacterial transformation | genetic exchange | recombinase loader | recombination mediator protein | horizontal gene transfer

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Section: Resultsmentioning

confidence: 99%

Structure–function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation

Quevillon‐Chéruel

Campo

Mirouze

et al. 2012

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

154

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“…The mutated MCM-BP was then subcloned between the XbaI and NotI sites in pMZS3F (Zeghouf et al, 2004) using standard PCR protocols. The negative control plasmid for rescue experiments was pMZS3F with lacZ cDNA inserted between the XhoI and NotI sites.…”

Section: Methodsmentioning

confidence: 99%

The MCM-associated protein MCM-BP is important for human nuclear morphology

Jagannathan

Sakwe

Nguyen

et al. 2012

Journal of Cell Science

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SummaryMini-chromosome maintenance complex-binding protein (MCM-BP) was discovered as a protein that is strongly associated with human MCM proteins, known to be crucial for DNA replication in providing DNA helicase activity. The Xenopus MCM-BP homologue appears to play a role in unloading MCM complexes from chromatin after DNA synthesis; however, the importance of MCM-BP and its functional contribution to human cells has been unclear. Here we show that depletion of MCM-BP by sustained expression of short hairpin RNA (shRNA) results in highly abnormal nuclear morphology and centrosome amplification. The abnormal nuclear morphology was not seen with depletion of other MCM proteins and was rescued with shRNA-resistant MCM-BP. MCM-BP depletion was also found to result in transient activation of the G2 checkpoint, slowed progression through G2 and increased replication protein A foci, indicative of replication stress. In addition, MCM-BP depletion led to increased cellular levels of MCM proteins throughout the cell cycle including soluble MCM pools. The results suggest that MCM-BP makes multiple contributions to human cells that are not limited to unloading of the MCM complex.

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“…The protein is predicted to contain an N-terminal TM helix (11,13), and previous subcellular localization experiments have shown that tagged derivatives partition with the membrane fraction and that the C terminus is in the cytoplasm (13). Our group has used the sequential peptide affinity (SPA) tag (14) to observe the accumulation of multiple sprotein fusions (11). Because AcrZ-SPA is easily detected (11) and a strain producing the tagged derivative is phenotypically wild type in a cell envelope stress assay (15) as well as in the antibiotic sensitivity assays described below, AcrZ-SPA was a good candidate for biochemical approaches to identify interacting protein partners.…”

Section: Acrz-spa Copurifies With Components Of the Acrab-tolc Membranementioning

confidence: 99%

Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance

Hobbs

Yin

Paul

et al. 2012

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

184

188

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The AcrAB-TolC multidrug efflux pump confers resistance to a wide variety of antibiotics and other compounds in Escherichia coli. Here we show that AcrZ (formerly named YbhT), a 49-amino-acid inner membrane protein, associates with the AcrAB-TolC complex. Copurification of AcrZ with AcrB, in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB. The highly conserved acrZ gene is coregulated with acrAB through induction by the MarA, Rob, and SoxS transcription regulators. In addition, mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC. This differential antibiotic sensitivity suggests that AcrZ may enhance the ability of the AcrAB-TolC pump to export certain classes of substrates.multidrug resistance | resistance-nodulation-division | RND superfamily

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Sequential Peptide Affinity (SPA) System for the Identification of Mammalian and Bacterial Protein Complexes

Cited by 191 publications

References 18 publications

Structure–function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation

Structure–function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation

The MCM-associated protein MCM-BP is important for human nuclear morphology

Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance

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