Escherichia coli YegI is a novel Ser/Thr kinase lacking conserved motifs that localizes to the inner membrane.
Ontology highlight
ABSTRACT:
SUBMITTER: Rajagopalan K
PROVIDER: S-EPMC7658054 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ?19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ?25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.
| S-EPMC3591654 | biostudies-literature
Project description:In bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys over the past decade in phylogenetically diverse bacteria have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes, such as that of Escherichia coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases, which have extensive sequence and structural homology to eukaryotic Ser/Thr protein phosphatase 2C (PP2C) phosphatases. These proteins, called eukaryote-like Ser/Thr phosphatases (eSTPs), have been identified in a number of bacteria but not in E. coli Here, we describe a previously unknown eSTP encoded by an E. coli open reading frame (ORF), yegK, and characterize its biochemical properties, including its kinetics, substrate specificity, and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely related E. coli strains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase that is encoded in the same operon.IMPORTANCE Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacterium Escherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported in E. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.
| S-EPMC6112009 | biostudies-literature
Project description:Protein interactions are essential for most cellular functions. Interactions mediated by domains that appear in a large number of proteins are of particular interest since they are expected to have an impact on diversities of cellular processes such as signal transduction and immune response. Many well represented domains recognize and bind to primary sequences less than 10 amino acids in length called Short Linear Motifs (SLiMs).In this study, we systematically studied the evolutionary conservation of SLiMs recognized by SH2, SH3 and Ser/Thr Kinase domains in both ordered and disordered protein regions. Disordered protein regions are protein sequences that lack a fixed three-dimensional structure under putatively native conditions. We find that, in all these domains examined, SLiMs are more conserved in disordered regions. This trend is more evident in those protein functional groups that are frequently reported to interact with specific domains.The correlation between SLiM conservation with disorder prediction demonstrates that functional SLiMs recognized by each domain occur more often in disordered as compared to structured regions of proteins.
| S-EPMC2559891 | biostudies-other
Project description:Gram-negative bacteria depend on energised protein complexes that connect the two membranes of the cell envelope. However, ?-barrel outer-membrane proteins (OMPs) and ?-helical inner-membrane proteins (IMPs) display quite different organisation. OMPs cluster into islands that restrict their lateral mobility, while IMPs generally diffuse throughout the cell. Here, using live cell imaging of Escherichia coli, we demonstrate that when transient, energy-dependent transmembrane connections are formed, IMPs become subjugated by the inherent organisation of OMPs and that such connections impact IMP function. We show that while establishing a translocon for import, the colicin ColE9 sequesters the IMPs of the proton motive force (PMF)-linked Tol-Pal complex into islands mirroring those of colicin-bound OMPs. Through this imposed organisation, the bacteriocin subverts the outer-membrane stabilising role of Tol-Pal, blocking its recruitment to cell division sites and slowing membrane constriction. The ordering of IMPs by OMPs via an energised inter-membrane bridge represents an emerging functional paradigm in cell envelope biology.
| S-EPMC5852019 | biostudies-literature
Project description:Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.
| S-EPMC3259860 | biostudies-other
Project description:The phospholipids of Escherichia coli consist mainly of phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin. PG makes up ?25% of the cellular phospholipid and is essential for growth in wild-type cells. PG is synthesized on the inner surface of the inner membrane from cytidine diphosphate-diacylglycerol and glycerol 3-phosphate, generating the precursor phosphatidylglycerol-phosphate (PGP). This compound is present at low levels (?0.1% of the total lipid). Dephosphorylation of PGP to PG is catalyzed by several PGP-phosphatases. The pgpA and pgpB genes, which encode structurally distinct PGP-phosphatases, were identified previously. Double deletion mutants lacking pgpA and pgpB are viable and still make PG, suggesting the presence of additional phosphatase(s). We have identified a third PGP-phosphatase gene (previously annotated as yfhB but renamed pgpC) using an expression cloning strategy. A mutant with deletions in all three phosphatase genes is not viable unless covered by a plasmid expressing either pgpA, pgpB, or pgpC. When the triple mutant is covered with the temperature-sensitive plasmid pMAK705 expressing any one of the three pgp genes, the cells grow at 30 but not 42 °C. As growth slows at 42 °C, PGP accumulates to high levels, and the PG content declines. PgpC orthologs are present in many other bacteria.
| S-EPMC3037664 | biostudies-literature
Project description:Nearly a quarter of the Escherichia coli genome encodes for inner membrane proteins of which approximately a third have unassigned or poorly understood function. We had previously demonstrated that the synergy between the functional roles of the inner membrane-spanning YciB and the inner membrane lipoprotein DcrB, is essential in maintaining cell envelope integrity. In yciB dcrB cells, the abundant outer membrane lipoprotein, Lpp, mislocalizes to the inner membrane where it forms toxic linkages to peptidoglycan. Here, we report that the aberrant localization of Lpp in this double mutant is due to inefficient lipid modification at the first step in lipoprotein maturation. Both Cpx and Rcs signaling systems are upregulated in response to the envelope stress. The phosphatidylglycerol-pre-prolipoprotein diacylglyceryl transferase, Lgt, catalyzes the initial step in lipoprotein maturation. Our results suggest that the attenuation in Lgt-mediated transacylation in the double mutant is not a consequence of lowered phosphatidylglycerol levels. Instead, we posit that altered membrane fluidity, perhaps due to changes in lipid homeostasis, may lead to the impairment in Lgt function. Consistent with this idea, a dcrB null is not viable when grown at low temperatures, conditions which impact membrane fluidity. Like the yciB dcrB double mutant, dcrB null-mediated toxicity can be overcome in distinct ways - by increased expression of Lgt, deletion of lpp, or removal of Lpp-peptidoglycan linkages. The last of these events leads to elevated membrane vesiculation and lipid loss, which may, in turn, impact membrane homeostasis in the double mutant.Importance A distinguishing feature of Gram-negative bacteria is their double-membraned cell envelope which presents a formidable barrier against environmental stress. In E. coli, more than a quarter of the cellular proteins reside at the inner membrane but about a third of these proteins are functionally unassigned or their function is incompletely understood. Here, we show that the synthetic lethality underlying the inactivation of two inner membrane proteins, a small integral membrane protein YciB, and a lipoprotein, DcrB, results from the attenuation of the first step of lipoprotein maturation at the inner membrane. We propose that these two inner membrane proteins YciB and DcrB play a role in membrane homeostasis in E. coli and related bacteria.
| S-EPMC8095458 | biostudies-literature
Project description:Understanding phosphorylation-mediated regulation of metabolic enzymes, pathways, and cell phenotypes under metabolic shifts represents a major challenge. The kinases associated with most phosphorylation sites and the link between phosphorylation and enzyme activity remain unknown. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteome and phosphoproteome analysis of Escherichia coli ΔyeaG, a strain lacking a poorly characterized serine/threonine kinase YeaG, to decipher kinase-substrate interactions and the effects on metabolic phenotype during shifts from glucose to malate. The starting point of our analysis was the identification of physiological conditions under which ΔyeaG exhibits a clear phenotype. By metabolic profiling, we discovered that ΔyeaG strain has a significantly shorter lag phase than the wild type during metabolic shift from glucose to malate. Under those conditions, our SILAC analysis revealed several proteins that were differentially phosphorylated in the ΔyeaG strain. By focusing on metabolic enzymes potentially involved in central carbon metabolism, we narrowed down our search for putative YeaG substrates and identified isocitrate lyase AceA as the direct substrate of YeaG. YeaG was capable of phosphorylating AceA in vitro only in the presence of malate, suggesting that this phosphorylation event is indeed relevant for glucose to malate shift. There is currently not enough evidence to firmly establish the exact mechanism of this newly observed regulatory phenomenon. However, our study clearly exemplifies the usefulness of SILAC-based approaches in identifying proteins kinase substrates, when applied in physiological conditions relevant for the activity of the protein kinase in question.
| S-EPMC8055822 | biostudies-literature
Project description:We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide.
| S-EPMC3910433 | biostudies-literature
Project description:How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.
| S-EPMC3326482 | biostudies-literature