Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex

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Novel Thioredoxin-Like Proteins Are Components of a Protein Complex Coating the Cortical Microtubules of Toxoplasma gondii

Microtubules are versatile biopolymers that support numerous vital cellular functions in eukaryotes. The specific properties of microtubules are dependent on distinct microtubule-associated proteins, as the tubulin subunits and microtubule structure are exceptionally conserved. Highly specialized microtubule-containing assemblies are often found in protists, which are rich sources for novel microtubule-associated proteins. A protozoan parasite, Toxoplasma gondii, possesses several distinct tubulin-containing structures, including 22 microtubules closely associated with the cortical membrane. Early ultrastructural studies have shown that the cortical microtubules are heavily decorated with associating proteins. However, little is known about the identities of these proteins. Here, we report the discovery of a novel protein, TrxL1 (for Thioredoxin-Like protein 1), and an associating complex that coats the cortical microtubules. TrxL1 contains a thioredoxin-like fold. To visualize its localization in live parasites by fluorescence, we replaced the endogenous TrxL1 gene with an mEmeraldFP-TrxL1 fusion gene. Structured illumination-based superresolution imaging of this parasite line produced a detailed view of the microtubule cytoskeleton. Despite its stable association with the cortical microtubules in the parasite, TrxL1 does not seem to bind to microtubules directly. Coimmunoprecipitation experiments showed that TrxL1 associates with a protein complex containing SPM1, a previously reported microtubule-associated protein in T. gondii. We also found that SPM1 recruits TrxL1 to the cortical microtubules. Besides SPM1, several other novel proteins are found in the TrxL1-containing complex, including TrxL2, a close homolog of TrxL1. Thus, our results reveal for the first time a microtubule-associated complex in T. gondii.     

Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome

Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.     

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 ⁴²⁰AKKAA⁴²⁴ mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gα_q association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.     

The Cholinergic Stimulating Effects of Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor Are Mediated by Protein Kinase C

Abstract: The intracellular mechanisms through which two trophic factors, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), regulate cholinergic development were examined in sympathetic neuron cultures. Treatment with CNTF or LIF increased levels of choline acetyltransferase (ChAT) activity by 375 and 350%, respectively. However, in neuronal cultures depleted of protein kinase C (PKC) activity by chronic phorbol ester treatment, neither CNTF nor LIF elevated ChAT activity. Further, the stimulation of ChAT due to increased cell density was not observed in PKC-depleted sympathetic neurons. The inhibition of CNTF-stimulated ChAT by phorbol ester occurred in a dose-dependent manner and chronic phorbol ester treatments did not alter the levels of the catecholamine biosynthetic enzyme tyrosine hydroxylase. Moreover, increased levels of diacylglycerol, an endogenous activator of PKC, were observed in sympathetic neurons treated with CNTF. However, neither CNTF nor LIF stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate. These observations suggest that a common PKC-dependent pathway, which is independent of phosphatidylinositol 4,5-bisphosphate hydrolysis, mediates the cholinergic stimulating effects of CNTF, LIF, and cell-cell contact in cultured sympathetic neurons.     

Rescue of PINK1 Protein Null-specific Mitochondrial Complex IV Deficits by Ginsenoside Re Activation of Nitric Oxide Signaling

PINK1, linked to familial Parkinson''s disease, is known to affect mitochondrial function. Here we identified a novel regulatory role of PINK1 in the maintenance of complex IV activity and characterized a novel mechanism by which NO signaling restored complex IV deficiency in PINK1 null dopaminergic neuronal cells. In PINK1 null cells, levels of specific chaperones, including Hsp60, leucine-rich pentatricopeptide repeat-containing (LRPPRC), and Hsp90, were severely decreased. LRPPRC and Hsp90 were found to act upstream of Hsp60 to regulate complex IV activity. Specifically, knockdown of Hsp60 resulted in a decrease in complex IV activity, whereas antagonistic inhibition of Hsp90 by 17-(allylamino) geldanamycin decreased both Hsp60 and complex IV activity. In contrast, overexpression of the PINK1-interacting factor LRPPRC augmented complex IV activity by up-regulating Hsp60. A similar recovery of complex IV activity was also induced by coexpression of Hsp90 and Hsp60. Drug screening identified ginsenoside Re as a compound capable of reversing the deficit in complex IV activity in PINK1 null cells through specific increases of LRPPRC, Hsp90, and Hsp60 levels. The pharmacological effects of ginsenoside Re could be reversed by treatment of the pan-NOS inhibitor l-NG-Nitroarginine Methyl Ester (l-NAME) and could also be reproduced by low-level NO treatment. These results suggest that PINK1 regulates complex IV activity via interactions with upstream regulators of Hsp60, such as LRPPRC and Hsp90. Furthermore, they demonstrate that treatment with ginsenoside Re enhances functioning of the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis in PINK1 null neurons by restoring NO levels, providing potential for new therapeutics targeting mitochondrial dysfunction in Parkinson''s disease.     

Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.     

WD Repeat Protein WDR48 in Complex with Deubiquitinase USP12 Suppresses Akt-dependent Cell Survival Signaling by Stabilizing PH Domain Leucine-rich Repeat Protein Phosphatase 1 (PHLPP1)

PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.     

Reconstitution of Plant Alkane Biosynthesis in Yeast Demonstrates That Arabidopsis ECERIFERUM1 and ECERIFERUM3 Are Core Components of a Very-Long-Chain Alkane Synthesis Complex

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.     

F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation

F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.The F plasmid (99,159 bp) of Escherichia coli is a model system for the study of the horizontal gene transfer among prokaryotes via conjugation (3, 10, 27). F encodes a 33.3-kb transfer region that is responsible for the formation of mating junctions between donor and recipient cells prior to DNA transfer and establishment in the recipient. The hallmark of F conjugation is the formation of extracellular filaments, F pili, that initiate contact between mating cells and retract, bringing the donor and recipient cells together (5, 19). Synthesis of the F pilus is not well understood, despite the morphological simplicity of this organelle (7, 15, 28). The F transfer region consists of nearly 40 tra genes, with 18 being involved in construction of the transferosome, which is involved in pilus synthesis, mating pair stabilization, and DNA transfer (9). Eight of the encoded Tra proteins (TraA, -B, -C, -E, -G [the N-terminal domain], -K, -L, and -V) correspond to widely conserved members of type IV secretion systems (T4SS), whereas another 9 (TraF, -G [C-terminal domain], -H, -N, -U, and -W and TrbB, -C, and -I) are involved in the F-specific T4SS (4, 18). Two other proteins (TraQ and -X) are specific to the F plasmid itself. The roles of the F-specific proteins that are involved in pilus assembly and DNA transfer are intriguing, since other conjugative T4SS appear to function efficiently without them (18). These tra proteins do not affect F pilin levels, and hence, they have been assigned functions in pilus assembly/retraction and mating pair stabilization, which are characteristics of F-like transfer systems (18). TraF, -H, and -W and TrbC are required for F pilus assembly (9), and mutations in traU reduce the number and the mean length of pili but do not abolish pilus outgrowth (24). TraU is required for DNA transfer and has been tentatively grouped with TraN and -G as proteins involved in mating pair stabilization (18). TrbI is thought to play a role in pilus retraction, since trbI mutants have unusually long pili (21). TrbB contains the thioredoxin-like domain with a C-X-X-C motif and appears to be a periplasmic disulfide bond isomerase (6). Previously, we hypothesized that TrbB and TraF, the latter of which also has the thioredoxin-like domain but lacks the C-X-X-C motif, might have chaperone-like activity. These proteins might help F T4SS proteins such as TraH, -U, and -N, which have 6, 10, and 22 conserved cysteines, respectively, achieve the correct conformation for assembly into the transferosome complex (6). Interestingly, yeast two-hybrid (Y2H) analysis demonstrated that TraF, -H, -U, and -W and TrbB and -I form an interaction group, with TraH directly linked to TraF, TraU, and TrbI (14). TraH is the only one of the three cysteine-rich proteins required for pilus assembly; it is the largest protein (458 amino acids [aa]; 50.2-kDa precursor, processed to 47.8 kDa) in the interaction group and contains a C-terminal coiled-coil domain that can contribute to its oligomerization and interaction with other T4SS proteins (18). Y2H analysis also showed that the C-terminal region of TraH is critical for its interaction with TraF (28 kDa, processed to 25.9 kDa) and TraU (36.8 kDa, processed to 34.3 kDa) and that a deletion within the N-terminal region of TraH enhanced its interaction with TrbI (14.1 kDa) (14).Mutations in traH affect pilus outgrowth but not pilus tip formation at the cell surface, since traH mutants are sensitive to the M13K07 transducing phage, which binds to the pilus tip (1). Membrane fractionation studies of cells containing subclones of the F transfer region originally suggested that TraH fractionates with the inner membrane (IM) (22). TraH contains three N-terminal hydrophobic domains of approximately 20 aa each, which supports this model. In contrast, Ham et al. predicted TraH to be a soluble periplasmic protein (12). Sucrose density gradient sedimentation studies suggested that FLAG-tagged TraH, in the presence of F lac traH80, is in the outer membrane (OM) (23). Since TraH is extracted from membrane preparations with guanidine-HCl or urea but not Triton X-100, Manwaring concluded that TraH is a peripherally associated outer membrane protein (23). By use of subclones of the F transfer region, TraF, -U, -W, and TrbB were localized to the periplasm, whereas TrbI was thought to be an inner membrane protein (21, 24, 29, 30). Using the F plasmid derivative pOX38-Tc (2), which carries the entire F transfer region, we reassessed the localization of TraH as well as TraF, TraU, and TraW (23.6 kDa, processed to 21.7 kDa).E. coli strains were grown at 37°C in Luria-Bertani (LB) broth (1% tryptone [Difco], 0.5% yeast extract [Difco], 1% NaCl [BDH]) with shaking to mid-exponential phase (optical density at 600 nm [OD₆₀₀] of ca. 0.5) with appropriate antibiotics at the following concentrations: 50 μg/ml ampicillin (Ap), 20 μg/ml chloramphenicol (Cm), 25 μg/ml kanamycin (Km), 200 μg/ml streptomycin (Sm), 100 μg/ml spectinomycin (Sp), and 10 μg/ml tetracycline (Tc). Sucrose density flotation studies of cell membrane fractions and immunoblot analysis were performed as previously described (17). Cell pellets corresponding to 0.1 OD₆₀₀ equivalents were used in all immunoblot assays. Samples were boiled in sodium dodecyl sulfate (SDS) sample buffer for 5 min and were analyzed by resolving SDS-15% polyacrylamide gel by using the Bio-Rad Minigel system. The positions of the inner and outer membrane fractions were determined using polyclonal antibodies to the C-terminal region of OmpA, the major outer membrane porin, and CpxA, the inner membrane sensor of the CpxAR two-component system (25). Anti-CpxA, anti-TraE, anti-TraF, anti-TraH, anti-TraU, anti-TraW, and anti-TrbB polyclonal antisera (raised in rabbits) were diluted 1:7,000, 1:5,000, 1:2,000, 1:1,000; 1:500, 1:20,000 and 1:10,000, respectively, in blocking solution and were incubated with the blots at room temperature for 1 h. Anti-OmpA antibodies were used at a 10⁻⁵ dilution in 5% bovine serum albumin (BSA; Roche) to avoid heavy background. Unfortunately, TrbI protein could not be overproduced and specific antibodies could not be raised.Log-phase cultures of E. coli MC4100 (Sm^r) (17) containing pOX38-Tc (2) were separated into periplasmic, cytoplasmic, and membrane fractions according to a previously described method (26). The fractions were tested for the presence of TraH, TraF, TraU, and TraW by SDS-PAGE, followed by immunoblot analysis. All four proteins were found associated with the membrane fraction and not the periplasmic fraction (Fig. ​(Fig.1A).1A). TrbB was found in the periplasmic fraction, in agreement with its proposed role in disulfide bond isomerization (6; data not shown).Open in a separate windowFIG. 1.(A) The F-specific proteins TraH, -F, -U, and -W were detected in the membrane fraction when expressed from MC4100/pOX38-Tc. Proteins were detected by immunoblotting using antisera specific for each protein as described in the text. (B) TraF localization was tested in pOX38-Km and pOX38 ΔtraV::cat. The cells were fractionated into cytoplasmic (C), periplasmic (P), and total membrane (M) fractions, and TraF was detected by immunoblotting with anti-TraF antibodies. TraV was complemented by pRS29 (pRS31 acted as a negative control). The following abbreviations are used: WT, wild type; ΔV, pOX38 ΔtraV::cat; ΔH, pOX38-Tc ΔtraH::cat; and ΔF, pOX38 ΔtraF::kan. The positions of the proteins are indicated by arrows on the right of each panel. The asterisk indicates a band that reacts nonspecifically with anti-F antiserum.Sucrose density flotation gradients of the membrane preparations of MC4100 (Sm^r) cells harboring pOX38-Tc (2), pOX38-Tc ΔtraF::kan (6) and pOX38-Tc ΔtraH::cat were performed to distinguish between OM and IM proteins according to reference 17. pOX38-Tc ΔtraH::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase cassette into traH. Gradients were fractionated, and a subset of the fractions (fractions 26 to 54, renamed 1 to 29) that contained the proteins of interest were subjected to SDS-PAGE and immunoblot analyses (Fig. ​(Fig.2).2). OmpA and CpxA were controls for the outer and inner membrane fractions and helped define the subset of fractions examined (Fig. ​(Fig.2,2, panels 1 and 2, respectively). The TraE pilus assembly protein of the F plasmid was used as an IM marker for the F transfer system (Fig. ​(Fig.2,2, panel 9) (9). TraH fractionated as an OM protein in MC4100/pOX38-Tc (Fig. ​(Fig.2,2, panel 3), as did TraF, TraU, and TraW (Fig. ​(Fig.2,2, panels 5, 7, and 8, respectively). TraH did not appear to be required for TraF localization, which was unaffected in a traH mutant (Fig. ​(Fig.2,2, panel 6). In addition, TraF did not appear to be required for TraH localization, although its absence caused a reduction in the levels of TraH (Fig. ​(Fig.2,2, panel 4; see below).Open in a separate windowFIG. 2.The cellular localizations of TraE, TraF, TraH, TraU, and TraW in subcellular fractions of E. coli MC4100/pOX38-Tc and its derivatives. Flotation sucrose density gradients were performed with subsequent immunodetection of tra proteins in a subset of gradient fractions (fractions 26 to 54, renumbered 1 to 29). The positions of the IM and OM fractions are shown above the gels, and the identities of the samples are indicated on the left. The panel numbers are indicated on the right.TraF, -H, -U, and -W appear to be periplasmic proteins that associate with the outer membrane when in the context of the complete transfer apparatus. TrbC, which is fused to TraW in the F-like R27 T4SS, might also be part of this complex (18). Therefore, an as yet unidentified transfer protein should act as an anchor in the outer membrane, directing these proteins to this location. Of the 18 transferosome proteins, only TraV and TraN are known to be located in the OM, with TraV being the only OM protein involved in pilus assembly. Preliminary localization studies using TraF as a test case and a traV insertion mutant, pOX38 ΔtraV::cat (this study, constructed as described above for pOX38-Tc ΔtraH::cat), demonstrated that the levels of TraF decreased dramatically. However, the remaining TraF was found in the periplasm (Fig. ​(Fig.1B).1B). Complementation of the traV mutation with pRS29, but not pRS31 (1), restored TraF localization to the outer membrane. Thus, TraV is probably the anchor protein for both the F-specific transferosome proteins (TraF, -H, -U, and -W) as well as the TraV, -K, and -B complex (13).MC4100 (Sm^r) cells bearing pOX38-Tc (2) or insertion mutant pOX38-Tc ΔtraH::cat, pOX38-Tc ΔtraF::kan (6), pOX38-Tc ΔtrbB::cat (6), pOX38-Tc ΔtraW::cat (this study), pOX38 traU347 (Km^r) (24), or pOX38-trbI472 (Km^r) (21) were used in subsequent experiments. pOX38-Tc ΔtraW::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase gene within traW. Mating efficiencies of these mutants were determined according to previously described methods using E. coli ED24 (Sp^r) as the recipient (20). Transconjugants were selected based on double resistance toward chloramphenicol or kanamycin and spectinomycin (Fig. ​(Fig.2).2). Observed mating efficiencies were in agreement with the data obtained previously, as were the results of complementation assays using subclones carrying the appropriate transfer gene (1, 6, 21, 24, 29, 30). These subclones were pK184TraH (Km^r) (this study), pFTraF and pFTrbB (Ap^r) (6), and pKI175 (Ap^r; traWU) (30) (Fig. ​(Fig.2).2). pK184TraH is based on the vector pK184 (Km^r) and contains the traH gene plus its ribosome binding site cloned into the EcoRI and HindIII sites in pK184 (16). Immunoblot analyses revealed that traF, traU, or trbB, but not traW, insertion mutants had slightly reduced levels of TraH in MC4100 cells whereas the trbI insertion mutant had undetectable levels of TraH (Fig. ​(Fig.3).3). Since TraH interacts directly with TrbI, TraF, and TraU in Y2H assays (14), the absence of these proteins would be expected to destabilize TraH. TraH is thought to interact indirectly with TraW via TraU (14); its levels were unaffected in a traW mutant. TraH was destabilized in a dsbA mutant and was undetectable by immunoblotting (data not shown) and decreased slightly in a trbB mutant, suggesting that disulfide bond formation (DsbA) and isomerization (TrbB) are important for TraH.Open in a separate windowFIG. 3.Immunoblot analysis of the levels of TraH in the absence of other members of Y2H interaction group by using pOX38-Tc and its derivatives containing insertion mutations in traH, traF, trbB, traW, traU, or trbI. A loading control is shown in the lower panel, and the mating efficiency (ME) expressed as a percentage of transconjugants relative to donor cells is given below the gels. n.d., not detected; n.a., not applicable. The last line of data are the complementation data (percent complementation mating efficiency [CME]) obtained by use of clones as described in the text. Previously, TraH was found to interact with TraF, TraU, and TrbI, and TraU interacts with TraW (14).The absence of TrbI appeared to have the most profound effect on the level of TraH, although there was only a 20-fold decrease in mating efficiency, suggesting that enough TraH was present to support mating (Fig. ​(Fig.3).3). Complementation assays performed with pOX38-trbI472 and pBAD24TrbI plasmids (this study) restored the levels of TraH, possibly by stabilizing it (Fig. ​(Fig.3).3). pBAD24TrbI is based on the vector pBAD24 (Ap^r) and contains the trbI gene cloned into the EcoRI site in pBAD24 (11). However, complementation with pBAD24TrbI did not restore mating efficiency to wild-type levels, confirming that the insertion mutation within pOX38-trbI472 has a weak polar effect on downstream genes in the tra operon (21). Alternatively, overexpression of TrbI from pBAD24TrbI affected mating efficiency.Y2H analysis revealed two regions within TraH that appeared to be important for TraH-TrbI interactions (14). The deletion of 50 N-terminal amino acids (aa 25 to 75) from the mature TraH gave a 40-fold increase in TraH-TrbI interaction in the Y2H assay (14). This region of TraH also contains the highly conserved residues N31, T44, G60, and R65 (numeration includes the 25-aa signal peptide) (Fig. ​(Fig.4A).4A). Site-directed mutagenesis was performed on plasmid pK184TraH by using the QuikChange kit (Stratagene). The mating abilities of MC4100/pOX38-Tc ΔtraH::cat/pK184TraH and derivatives with amino acid substitutions N31A, T44A, G60A, and R65A were determined according to previously described methods using ED24 (Sp^r) as the recipient (20). Transconjugants were selected based on double resistance toward tetracycline and spectinomycin. TraH levels within the donor cells were monitored by immunoblot analysis. The N31A and T44A substitutions did not affect mating efficiency and did not change the level of TraH within donor cells (Fig. ​(Fig.5).5). The G60A and R65A substitutions decreased mating efficiency to undetectable levels. TraH levels remained unchanged in both mutants (Fig. ​(Fig.5).5). MC4100/pOX38-Tc ΔtraH::cat cells with pK184TraHG60A or pK184TraHR65A were also resistant toward pilus-specific phage f1, suggesting that the pilus was not assembled.Open in a separate windowFIG. 4.Multiple sequence alignment of F-like TraH proteins. (A) Alignment of the N-terminal regions. (B) Alignment of the putative TraH-TrbI interaction region. The leader peptide is cleaved after A24 in F TraH, which is marked by an arrow. The degrees of identity are indicated by black and gray boxes above the sequences, with the tallest black boxes representing conservation over all 7 sequences. Positions with 5 or more different amino acids are marked with the shortest black boxes. The gray boxes in the residue number line indicate gaps in some of the sequences. The putative nucleotide triphosphate (NTP) binding site (aa 193 to 200) and the conserved sequence (aa 220 to 226) are underlined. The regions thought to interact with TrbI are bracketed. Asterisks refer to amino acids selected for mutational analysis. GenBank protein accession numbers for the sequences are as follows: for F, {"type":"entrez-protein","attrs":{"text":"BAA97968","term_id":"8918921","term_text":"BAA97968"}}BAA97968; for SXT, {"type":"entrez-protein","attrs":{"text":"AAL59676","term_id":"21885270","term_text":"AAL59676"}}AAL59676; for R391, {"type":"entrez-protein","attrs":{"text":"AAM08008","term_id":"20095142","term_text":"AAM08008"}}AAM08008; for pNL1, {"type":"entrez-protein","attrs":{"text":"NP_049152","term_id":"10956932","term_text":"NP_049152"}}NP_049152; for RTS1, {"type":"entrez-protein","attrs":{"text":"NP_640201","term_id":"21233903","term_text":"NP_640201"}}NP_640201; for pED208, {"type":"entrez-protein","attrs":{"text":"AAM90722","term_id":"22539465","term_text":"AAM90722"}}AAM90722; and for R27, {"type":"entrez-protein","attrs":{"text":"NP_058340","term_id":"10957316","term_text":"NP_058340"}}NP_058340. Sequence alignment was performed with DNAStar software (LazerGene), using the ClustalW algorithm. Highly conserved amino acids as well as a consensus sequence are given above the residue number line.Open in a separate windowFIG. 5.Immunoblot analysis of intracellular levels of TraH in MC4100/pOX38-Tc ΔtraH::cat complemented with different pK184TraH plasmids. C represents the vector control pK184, WT is the wild-type pK184TraH plasmid, and an asterisk refers to the nonspecific band used as the loading control. Mating efficiency (ME), expressed as a percentage of transconjugants relative to donor cells, is given below the gels. n.d., not detected; MW, molecular mass.Sequence analysis also showed the presence of conserved residues N(L/I/Y)X(W/Y)XX(F/L) (N₂₂₀IMWNAL₂₂₆ in F TraH) within the putative TrbI interaction domain (aa 193 to 226) (Fig. ​(Fig.4B)4B) (14). Substitution of N220 with alanine (N220A) did not change the levels of TraH protein in pOX38-Tc ΔtraH::cat/pK184TraHN220A but decreased the mating ability to undetectable levels. The W223A mutation in TraH decreased the level of TraH within donor cells and reduced the mating efficiency 1,000-fold compared to the wild-type level (Fig. ​(Fig.5).5). The N220A and W223A mutants were resistant to f1 phage and could not assemble functional pili. Thus, mutations in N220 and W223 could affect TraH-TrbI interaction, or they may act independently to block TraH function. If TrbI is in the IM as previously reported (21), then the TrbI:TraH pair could be part of a second envelope-spanning structure analogous to the TraV:TraK:TraB scaffold (8, 13).Primary sequence analysis also revealed the presence of a putative Walker A motif within aa 193 to 226 of TraH (G₁₉₃CTVGGKS₂₀₀) (9). Comparison of seven TraH orthologs revealed that this motif is not conserved among TraH-like sequences (Fig. ​(Fig.4B).4B). To confirm whether this sequence might be important in the F plasmid, a triple mutant (G193A/K199A/S200A) was constructed. It reduced mating efficiency 20-fold but did not change the levels of TraH within donor cells (data not shown). Single substitutions (G193A, K199A, or S200A) did not change the mating efficiency or the level of TraH (data not shown). Thus, TraH, a peripheral OM protein, is probably not an NTPase, nor does it bind nucleotides.Our data also revealed that several conserved amino acid residues are critical for TraH function and structure and that TraH stability is dependent on TrbI as well as DsbA and TrbB, which affect disulfide bond formation and isomerization, respectively. Thus, TrbI, in which mutations have only a minor effect on mating ability, plays a more important role than previously thought (21).