Daniel O. Daley

A scalable, GFP-based pipeline for membrane protein overexpression screening and purification

We describe a generic, GFP‐based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane‐integral flavocytochrome. The approach is scalable and suitable for high‐throughput applications. The GFP‐based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.

Genes for Two Mitochondrial Ribosomal Proteins in Flowering Plants Are Derived from Their Chloroplast or Cytosolic Counterparts

Often during flowering plant evolution, ribosomal protein genes have been lost from the mitochondrion and transferred to the nucleus. Here, we show that substitution by a duplicated, divergent gene originally encoding the chloroplast or cytosolic ribosomal protein counterpart accounts for two missing mitochondrial genes in diverse angiosperms. The rps13 gene is missing from the mitochondrial genome of many rosids, and a transferred copy of this gene is not evident in the nucleus of Arabidopsis, soybean, or cotton. Instead, these rosids contain a divergent nuclear copy of an rps13 gene of chloroplast origin. The product of this gene from all three rosids was shown to be imported into isolated mitochondria but not into chloroplasts. The rps8 gene is missing from the mitochondrion and nucleus of all angiosperms examined. A divergent copy of the gene encoding its cytosolic counterpart (rps15A) was identified in the nucleus of four angiosperms and one gymnosperm. The product of this gene from Arabidopsis and tomato was imported successfully into mitochondria. We infer that rps13 was lost from the mitochondrial genome and substituted by a duplicated nuclear gene of chloroplast origin early in rosid evolution, whereas rps8 loss and substitution by a gene of nuclear/cytosolic origin occurred much earlier, in a common ancestor of angiosperms and gymnosperms.

Experimentally based topology models for E. coli inner membrane proteins

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a proteins C terminus and topology prediction. Here, we show that determination of the location of a proteins C terminus, rather than some internal loop, is the best strategy for large‐scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome‐scale studies.

Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase.

Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2.

Why genes persist in organelle genomes

Mitochondria and plastids (including chloroplasts) have a small but vital genetic coding capacity, but what are the properties of some genes that dictate that they must remain encoded in organelles?

Disassembly of the divisome in Escherichia coli: evidence that FtsZ dissociates before compartmentalization

In most bacteria cell division is mediated by a protein super‐complex called the divisome that co‐ordinates the constriction and scission of the cell envelope. FtsZ is the first of the divisome proteins to accumulate at the division site and is widely thought to function as a force generator that constricts the cell envelope. In this study we have used a combination of confocal fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) to determine if divisome proteins are present at the septum at the time of cytoplasmic compartmentalization in Escherichia coli. Our data suggest that many are, but that FtsZ and ZapA disassemble before the cytoplasm is sealed by constriction of the inner membrane. This observation implies that FtsZ cannot be a force generator during the final stage(s) of envelope constriction in E. coli.

Membrane protein structural biology - How far can the bugs take us? (Review)

Membrane proteins are core components of many essential cellular processes, and high-resolution structural data is therefore highly sought after. However, owing to the many bottlenecks associated with membrane protein crystallization, progress has been slow. One major problem is our inability to obtain sufficient quantities of membrane proteins for crystallization trials. Traditionally, membrane proteins have been isolated from natural sources, or for prokaryotic proteins, expressed by recombinant techniques. We are however a long way away from a streamlined overproduction of eukaryotic proteins. With this technical limitation in mind, we have probed the question as to how far prokaryotic homologues can take us towards a structural understanding of the eukaryotic/human membrane proteome(s).

Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.

The assembly of membrane proteins into complexes.

Protein complexes are a fundamental aspect of life in a membrane. It is therefore important to understand which proteins are assembled, and how the process of assembly is coordinated. To this end, a number of themes have emerged from the literature in recent years: first, membrane proteins assemble in an ordered, rather than a stochastic manner; second, they require chaperones to prevent unwanted interactions/aggregation; and third, they can be assembled into existing complexes. As these recurrent themes have emerged from studies on disparate complexes, they provide a general framework to understand the assembly of membrane proteins.

Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers

Background: EmrE is a dual-topology membrane protein, but it is not clear whether the active form is composed of parallel or anti-parallel dimers. Results: Antiparallel EmrE dimers are more stable than parallel dimers. Conclusion: Antiparallel EmrE dimers most likely correspond to the functional form of the protein. Significance: The results provide new information on the functional form of EmrE. The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.