Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.

The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions.     

An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins

Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.     

Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes

Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-length product only in four of the five coding sequences tested. A unique pattern of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing.     

High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.     

GALLEX,a measurement of heterologous association of transmembrane helices in a biological membrane

Whereas a variety of two-hybrid systems are available to measure the interaction of soluble proteins, related methods are significantly less developed for the measurement of membrane protein interactions. Here we present a two-hybrid system to follow the heterodimerization of membrane proteins in the Escherichia coli inner membrane. The method is based on the repression of a reporter gene activity by two LexA DNA binding domains with different DNA binding specificities. When coupled to transmembrane domains, heterodimeric association is reported by repression of beta-galactosidase synthesis. The LexA-transmembrane chimeric proteins were found to correctly insert into the membrane, and reproducible signals were obtained measuring the homodimerization as well as heterodimerization of wild-type and mutant glycophorin A transmembrane helices. The GALLEX data were compared with data recently gained by other methods and discussed in the general context of heteroassociation of single TM helices. Additionally, the formation of heterodimers between the TM domains of the alpha(4) and the beta(7) integrin subunits were tested. The results show that both homo- and heterodimerization of membrane proteins can be measured accurately using the GALLEX system.     

Use of yeast two-hybrid system for detection ofBacillus subtilis FtsZ protein partners

Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions. Three plasmids (pGBR1, pGBR2, pGBR3) with theClaI restriction site shifted in the three possible reading frames in fusion withGAL4 activating domain were constructed. The modified plasmids were used for identification of protein partners of FtsZ fromBacillus subtilis. Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified. The protein interactions were quantified by measurements of β-galactosidase activity in yeast cells using 4-methylumbelliferyl β-d-glactopyranoside as fluorogenic substrate.     

Prediction,assessment and validation of protein interaction maps in bacteria

High-throughput proteomics technologies, especially the yeast two-hybrid system, produce large volumes of protein-protein interaction data organized in networks. The complete sequencing of many genomes raises questions about the extent to which such networks can be transferred between organisms. We attempted to answer this question using the experimentally derived Helicobacter pylori interaction map and the recently described interacting domain profile pair (IDPP) method to predict a virtual map for Escherichia coli. The extensive literature concerning E.coli was used to assess all predicted interactions and to validate the IDPP method, which clusters protein domains by sequence and connectivity similarities. The IDPP method has a much better heuristic value than methods solely based on protein homology. The IDPP method was further applied to Campylobacter jejuni to generate a virtual interaction map. An in-depth comparison of the chemotaxis pathways predicted in E.coli and C.jejuni led to the proposition of new functional assignments. Finally, the prediction of protein-protein interaction maps across organisms enabled us to validate some of the interactions on the original experimental map.     

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

Protein diversity confers specificity in plasmid segregation

The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.     

Complex formation between recombinant ATP sulfurylase and APS reductase of Allium cepa (L.)

Recombinant ATP sulfurylase (AcATPS1) and adenosine-5'-phosphosulfate reductase (AcAPR1) from Allium cepa have been used to determine if these enzymes form protein-protein complexes in vitro. Using a solid phase binding assay, AcAPR1 was shown to interact with AcATPS1. The AcAPR1 enzyme was also expressed in E. coli as the N-terminal reductase domain (AcAPR1-N) and the C-terminal glutaredoxin domain (AcAPR1-C), but neither of these truncated proteins interacted with AcATPS1. The solid-phase interactions were confirmed by immune-precipitation, where anti-AcATPS1 IgG precipitated the full-length AcAPR1 protein, but not AcAPR1-N and AcAPR1-C. Finally, using the ligand binding assay, full-length AcATPS1 has been shown to bind to membrane-localised full-length AcAPR1. The significance of an interaction between chloroplastidic ATPS and APR in A. cepa is evaluated with respect to the control of the reductive assimilation of sulfate.     

Surface plasmon resonance imaging analysis of protein-protein interactions using on-chip-expressed capture protein

A surface plasmon resonance (SPR) imaging system, combined with a microwell gold chip for on-chip cell cultivation, was used to monitor protein-protein interactions. In particular, we developed an on-chip microscale cell cultivation system that integrates cell culture and on-chip analysis of protein-protein interactions on a single microwell chip in a time- and labor-saving manner. To assess the performance of this system in the analysis of protein-protein interactions, we conducted a series of protein-protein interaction analyses by measuring the binding of the yeast GAL4 dimerization domain (GAL4DD) to the GAL11 protein (GAL11P). Our system was found to enable the simple and rapid analysis of protein-protein interactions, requiring no special cell culturing equipment or recombinant protein expression prior to the immobilization of the purified proteins onto the chip. Our results demonstrate that the combination of an on-chip cell cultivation system and an SPR imaging system can be a useful tool to study protein-protein interactions without the need for time-consuming and labor-intensive protein preparation steps as well as fluorescent or other labeling of the interactants.