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Crystal structures of the CorA Mg(2+) channel have suggested that metal binding in the cytoplasmic domain stabilizes the pentamer in a closed conformation. The open "metal free" state of the channel is, however, still structurally uncharacterized. Here, we have attempted to map conformational states of CorA from Thermotoga maritima by determining which residues support the pentameric structure in the presence or absence of Mg(2+). We find that when Mg(2+) is present, the pentamer is stabilized by the putative gating sites (M1/M2) in the cytoplasmic domain. Strikingly however, we find that the conserved and functionally important periplasmic loop is vital for the integrity of the pentamer when Mg(2+) is absent from the M1/M2 sites. Thus, although the periplasmic loops were largely disordered in the x-ray structures of the closed channel, our data suggests a prominent role for the loops in stabilizing the open conformation of the CorA channels.

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.

Proteins embedded in membranes are important for helping the cell adapt to changes in the extracellular milieu and often play key roles in the life cycles of pathogenic microbes. Bioinformatic predictions can provide an estimate of membrane proteins, but experimental approaches of detection are required for a deeper understanding of their functions. To determine the effectiveness of experimental detection approaches, here we collate and discuss data from available proteomic analyses on the inner (or cytoplasmic) membrane of Escherichia coli. We compile a list of proteins that have been experimentally detected and by comparing this to a predicted proteome we identify membrane proteins that have eluded us experimentally. Limitations of current proteomic analyses together with possible solutions are discussed. We also provide a list of proteins for benchmarking the performance of future proteomic studies.

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.

The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis and structural studies. As a result, membrane proteomes remain ill-defined. Here, we report a global topology analysis of the Escherichia coli inner membrane proteome. Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, we derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. We also estimated the overexpression potential for 397 green fluorescent protein fusions; the results suggest that a large fraction of all inner membrane proteins can be produced in sufficient quantities for biochemical and structural work.

Gene expression for nuclear- and mitochondrial-encoded subunits of respiratory chain components was measured in developing soybean cotyledons and compared to the abundance of the relevant proteins. Overall respiratory gene expression peaked at day 16, close to the peak in cytochrome chain and TCA cycle activities from day 10 to 15. Protein abundance followed transcript abundance for all components examined with the exception of the F1beta subunit of ATP synthase. A dramatic peak in F1beta transcript levels early in development (day 5 to 7) was not mirrored by an increase in protein suggesting translational or post-translational control. Mitochondrial-encoded transcripts were at least 10-fold more abundant than nuclear-encoded transcripts. The pattern of transcript and protein abundance for uncoupling proteins displayed a trend similar to other respiratory proteins examined, implicating similar control mechanisms. The expression of alternative oxidase differed, increasing throughout development with protein peaking at day 20, perhaps suggesting a role in senescence. Overall, this study indicated that respiratory gene expression and protein abundance is co-ordinated with respiratory activity for most components but that some components, such as the F1beta subunit may be under discrete forms of regulation.

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.

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.