Interaction of the Escherichia coli transporter DctA with the sensor kinase DcuS: presence of functional DctA/DcuS sensor units

The aerobic Escherichia coli C(4) -dicarboxylate transporter DctA and the anaerobic fumarate/succinate antiporter DcuB function as obligate co-sensors of the fumarate responsive sensor kinase DcuS under aerobic or anaerobic conditions respectively. Overproduction under anaerobic conditions allowed DctA to replace DcuB in co-sensing, indicating their functional equivalence in this capacity. In vivo interaction studies between DctA and DcuS using FRET or a bacterial two-hybrid system (BACTH) demonstrated their interaction. DctA-YFP bound to an affinity column and was able to retain DcuS. DctA shows substantial sequence and secondary structure conservation to Glt(Ph) , the Na(+) /glutamate symporter of Pyrococcus horikoshii with known 3D structure. Topology studies of DctA demonstrated the presence of eight transmembrane helices in an arrangement similar to that of Glt(Ph) . DctA contains an additional predicted amphipathic helix 8b on the cytoplasmic side of the membrane that is specific for DctA and not present in Glt(Ph) . Mutational analysis demonstrated the importance of helix 8b in co-sensing and interaction with DcuS, and the isolated helix 8b showed strong interaction with DcuS. In DcuS, deletion and mutation of the cytoplasmic PAS(C) domain affected the interaction between DctA and DcuS. It is concluded that DctA forms a functional unit or sensor complex with DcuS through specific interaction sites.     

The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady‐state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.     

Distribution of the SELMA Translocon in Secondary Plastids of Red Algal Origin and Predicted Uncoupling of Ubiquitin-Dependent Translocation from Degradation

Protein import into complex plastids of red algal origin is a multistep process including translocons of different evolutionary origins. The symbiont-derived ERAD-like machinery (SELMA), shown to be of red algal origin, is proposed to be the transport system for preprotein import across the periplastidal membrane of heterokontophytes, haptophytes, cryptophytes, and apicomplexans. In contrast to the canonical endoplasmic reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to be uncoupled from proteasomal degradation. We investigated the distribution of known and newly identified SELMA components in organisms with complex plastids of red algal origin by intensive data mining, thereby defining a set of core components present in all examined organisms. These include putative pore-forming components, a ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was expanded. These newly identified putative SELMA components, as well as proteasomal subunits, were in vivo localized as PPC proteins in the diatom Phaeodactylum tricornutum. The presented data allow us to speculate about the specific features of SELMA translocation in contrast to the canonical ERAD system, especially the uncoupling of translocation from degradation.     

Transposons in biotechnologically relevant strains of Aspergillus niger and Penicillium chrysogenum

In the past 15 years, many class I and class II transposons were identified in filamentous fungi. However, little is known about the influence of transposons during industrial strain development. The availability of the complete genome sequences of the industrially relevant fungi Aspergillus niger and Penicillium chrysogenum has enabled an analysis of transposons present in these two fungi. Here, a compilation of the transposon-like sequences identified is provided. We investigated a yet undescribed A. niger retrotransposon, ANiTa1, as well as two P. chrysogenum transposons (PeTra1 and PeTra2), which are the first P. chrysogenum transposons ever described, in more detail. Analysis of the genomic distribution of selected transposable elements in five strains of A. niger and seven strains of P. chrysogenum revealed the transposon distribution to be virtually identical. However, one element, Vader-previously published-from A. niger, showed strain-specific differences in transposon distribution, suggesting transposition activity during classical strain improvement programs.     

Yersinia protein kinase YopO is activated by a novel G-actin binding process

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.     

Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.     

First selective agonist of the neuropeptide Y1‐receptor with reduced size

Selective NPY analogues are potent tools for tumour targeting. Their Y₁‐receptors are significantly over‐expressed in human breast tumours, whereas normal breast tissue only expresses Y₂‐receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y₁‐receptor agonists enhance the tumour to background ratio compared to Y₁‐receptor antagonists, we were interested in the development of Y₁‐receptor selective agonists. We designed 19 peptides containing the C‐terminus of NPY (28–36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y₁‐receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N‐terminally 5(6)‐carboxyfluorescein (CF) labelled peptides resulted in half‐lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y₁‐receptor internalization. For the first time, here we show the design and characterization of a small Y₁‐receptor selective agonist. This agonist might be a useful novel ligand for NPY‐mediated tumour diagnostics and therapeutics. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Biochemical Evidence for a Timing Mechanism in Prochlorococcus

Organisms coordinate biological activities into daily cycles using an internal circadian clock. The circadian oscillator proteins KaiA, KaiB, and KaiC are widely believed to underlie 24-h oscillations of gene expression in cyanobacteria. However, a group of very abundant cyanobacteria, namely, marine Prochlorococcus species, lost the third oscillator component, KaiA, during evolution. We demonstrate here that the remaining Kai proteins fulfill their known biochemical functions, although KaiC is hyperphosphorylated by default in this system. These data provide biochemical support for the observed evolutionary reduction of the clock locus in Prochlorococcus and are consistent with a model in which a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus is sufficient for biological timing in the very stable environment that Prochlorococcus inhabits.Cyanobacteria are photosynthetic prokaryotes that are known to possess a true circadian clock. Gene expression and other biological activities follow rhythmic cycles with a circa 24-h period. Rhythmic behavior is maintained even in the absence of environmental stimuli such as light and temperature. The underlying core oscillator consisting of the clock proteins KaiA, KaiB, and KaiC is the only characterized prokaryotic circadian oscillator. It was previously demonstrated that these three proteins, together with ATP, can produce 24-h oscillations of KaiC phosphorylation in vitro (17). The essential roles of KaiA and KaiB in oppositely influencing KaiC phosphorylation are well documented for the oscillator of “Synechococcus elongatus” PCC 7942 (hereafter S. elongatus), the species for which most bacterial circadian research has been conducted. Thus, it is puzzling that marine cyanobacteria of the genus Prochlorococcus, probably the most abundant photosynthetic organisms on Earth (5, 28), contain homologs of only two of these clock proteins, KaiC and KaiB (3, 11, 20). Laboratory cultures (10) as well as natural Prochlorococcus populations (24) display a rhythmic cell cycle together with a daily periodicity of gene expression that can be explained by the functioning of a circadian clock. Alternatively, these rhythms could be controlled directly by the daylight (10). The functional role of the Kai proteins from Prochlorococcus has remained entirely unclear and has not been experimentally addressed thus far.In the well-studied protein clock of S. elongatus, KaiC hexamers are at the center of the circadian oscillator, combining three intrinsic enzymatic activities: autokinase, autophosphatase, and ATPase. KaiA and KaiB modulate KaiC''s activities in opposite manners. KaiA seems to be essential for the shift between autophosphatase and autokinase, and for generating KaiC phosphorylation rhythms, by stabilizing C-terminal residues of KaiC, the A-loops (12). Thus, the absence of KaiA should have consequences for the enzymatic activities of the remaining Kai proteins of Prochlorococcus. In this study, the previously unknown functions of the Prochlorococcus sp. strain MED4 protein KaiB (ProKaiB) and ProKaiC are examined. In our in vitro experiments, we analyzed the recombinant proteins ProKaiB and ProKaiC in direct comparison to the core oscillator of S. elongatus, which consists of S. elongatus KaiA (SynKaiA), SynKaiB, and SynKaiC. We show here that both clock proteins from Prochlorococcus sp. strain MED4 independently exhibit their known biochemical functions, although the influence of ProKaiB on ProKaiC dephosphorylation is different certainly due to the absence of KaiA, the third protein of the oscillator. For ProKaiC, we demonstrate ATPase activity as well as the phosphorylation of serine 427 (S427) and threonine 428 (T428) using mass spectrometry and high-resolution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Moreover, we suggest that the deletion of kaiA is compensated by the enhanced autophosphorylation activity of ProKaiC. Our results might have further implications for the analysis of a possible timing mechanism in other bacterial species, such as purple bacteria that encode KaiB and KaiC homologs but that lack the KaiA component.