Distinct sensitivities of OmpF and PhoE porins to charged modulators.
(17/18963)
The inhibition of the anion-selective PhoE porin by ATP and of the cation-selective OmpF porin by polyamines has been previously documented. In the present study, we have extended the comparison of the inhibitor-porin pairs by investigating the effect of anions (ATP and aspartate) and positively charged polyamines (spermine and cadaverine) on both OmpF and PhoE with the patch-clamp technique, and by comparing directly the gating kinetics of the channels modulated by their respective substrates. The novel findings reported here are (1) that the activity of PhoE is completely unaffected by polyamines, and (2) that the kinetic changes induced by ATP on PhoE or polyamines on OmpF suggest different mechanisms of inhibition. ATP induces a high degree of flickering in the PhoE-mediated current and appears to behave as a blocker of ion flow during its presumed transport through PhoE. Polyamines modulate the kinetics of openings and closings of OmpF, in addition to promoting a blocker-like flickering activity. The strong correlation between sensitivity to inhibitors and ion selectivity suggests that some common molecular determinants are involved in these two properties and is in agreement with the hypothesis that polyamines bind inside the pore of cationic porins. (+info)
Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli.
(18/18963)
Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP. (+info)
MalK forms a dimer independent of its assembly into the MalFGK2 ATP-binding cassette transporter of Escherichia coli.
(19/18963)
The maltose transport complex (MTC) is a member of the ATP-binding cassette superfamily of membrane transport proteins and is a model for understanding the folding and assembly of hetero-oligomeric membrane protein complexes. The MTC is made up of two integral membrane proteins, MalF and MalG, and a peripheral membrane protein, MalK. These proteins associate with a stoichiometry of 1:1:2 to form the complex MalFGK2. In our studies of the oligomerization of this complex, we have shown that the ATP-binding component, MalK, forms a dimer in the absence of MalF and MalG. Epitope-tagged MalK coimmunoprecipitated with wild-type MalK, indicating that the MalK protein forms an oligomer. The relative amounts of tagged and wild-type MalK that were present in the whole cell extracts and in the immunoprecipitated complexes show that the MalK oligomer is a dimer. These hetero-oligomers can also be formed in vitro by mixing two extracts, each containing either tagged or wild-type MalK. The dimerization of MalK was also demonstrated in vivo using the bacteriophage lambda repressor fusion assay. The formation of a MalK dimer in the absence of MalF and MalG may represent an initial step in the assembly pathway of the MTC. (+info)
The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer.
(20/18963)
Germline mutations in two human mismatch repair (MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70% of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Although the hMLH1 protein has been found to copurify with another MMR protein hPMS2 as a heterodimer, their function in MMR is unknown. In this study, we have identified the physical interaction regions of both hMLH1 with hPMS2. We then examined the effects of hMLH1 missense alterations found in HNPCC kindreds for their interaction with hPMS2. Four of these missense alterations (L574P, K616Delta, R659P, and A681T) displayed >95% reduction in binding to hPMS2. Two additional missense alterations (K618A and K618T) displayed a >85% reduction in binding to hPMS2, whereas three missense alterations (S44F, V506A, and E578G) displayed 25-65% reduction in binding to hPMS2. Interestingly, two HNPCC missense alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1. These data confirm that functional deficiencies in the interaction of hMLH1 with hPMS2 are associated with HNPCC as well as suggest that other unknown functional alteration of the human MutL homologues may lead to tumorigenesis in HNPCC kindreds. (+info)
Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli.
(21/18963)
Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization. Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex. The dissociation constant was determined to be about 10(-6) M. The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli. (+info)
Xer site-specific recombination. DNA strand rejoining by recombinase XerC.
(22/18963)
Xer site-specific recombination functions in the stable maintenance of circular replicons in Escherichia coli. Each of two related recombinase proteins, XerC and XerD, cleaves a specific pair of DNA strands, exchanges them, and rejoins them to the partner DNA molecule during a complete recombination reaction. The rejoining activity of recombinase XerC has been analyzed using isolated covalent XerC-DNA complexes resulting from DNA cleavage reactions upon Holliday junction substrates. These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that covalently bound XerC can catalyze strand rejoining in the absence of other proteins. This contrasts with a recombinase-mediated cleavage reaction, which requires the presence of both recombinases, the recombinase mediating catalysis at any given time requiring activation by the partner recombinase. In a recombining nucleoprotein complex, both cleavage and rejoining can occur prior to dissociation of the complex. (+info)
An artificial transmembrane segment directs SecA, SecB, and electrochemical potential-dependent translocation of a long amino-terminal tail.
(23/18963)
Many integral membrane proteins contain an amino-terminal segment, often referred to as an N-tail, that is translocated across a membrane. In many cases, translocation of the N-tail is initiated by a cleavable, amino-terminal signal peptide. For N-tail proteins lacking a signal peptide, translocation is initiated by a transmembrane segment that is carboxyl to the translocated segment. The mechanism of membrane translocation of these segments, although poorly understood, has been reported to be independent of the protein secretion machinery. In contrast, here we describe alkaline phosphatase mutants containing artificial transmembrane segments that demonstrate that translocation of a long N-tail across the membrane is dependent upon SecA, SecB, and the electrochemical potential in the absence of a signal peptide. The corresponding mutants containing signal peptides also use the secretion machinery but are less sensitive to inhibition of its components. We present evidence that inhibition of SecA by sodium azide is incomplete even at high concentrations of inhibitor, which suggests why SecA-dependent translocation may not have been detected in other systems. Furthermore, by varying the charge around the transmembrane segment, we find that in the absence of a signal peptide, the orientation of the membrane-bound alkaline phosphatase is dictated by the positive inside rule. However, the presence of a signal peptide is an overriding factor in membrane orientation and renders all mutants in an Nout-Cin orientation. (+info)
Rapid modification of bacterial artificial chromosomes by ET-recombination.
(24/18963)
We present a method to modify bacterial artificial chromosomes (BACs) resident in their host strain. The method is based on homologous recombination by ET-cloning. We have successfully modified BACs at two distinct loci by recombination with a PCR product containing homology arms of 50 nt. The procedure we describe here is rapid, was found to work with high efficiency and should be applicable to any BAC modification desired. (+info)