Nearest neighbor analysis of the SecYEG complex. 1. Identification of a SecY-SecG interface

Integral membrane components SecY, SecE, and SecG of protein translocase form a complex in the Escherichia coli plasma membrane. To characterize subunit interactions of the SecYEG complex, a series of SecY variants having a single cysteine in its cytoplasmic (C1-C6) or periplasmic (P1-P5) domain were subjected to site-specific cross-linking experiments using bifunctional agents with thiol-amine reactivity. Experiments using inverted membrane vesicles revealed specific cross-linkings between a cysteine residue placed in the C2 or C3 domain of SecY and the cytosolic lysine (Lys26) near the first transmembrane segment of SecG. These SecY Cys residues also formed a disulfide bond with an engineered cytosolic cysteine at position 28 of SecG. Thus, the C2-C3 region of SecY is in the proximity of the N-terminal half of the SecG cytoplasmic loop. Experiments using spheroplasts revealed the physical proximity of P2 (SecY) and the C-terminal periplasmic region of SecG. In addition, mutations in secG were isolated as suppressors against a cold-sensitive mutation (secY104) affecting the TM4-C3 boundary of SecY. These results collectively suggest that a C2-TM3-P2-TM4-C3 region of SecY serves as an interface with SecG.     

Nearest neighbor nucleotide patterns. Structural and biological implications

Recently, nearest neighbor patterns were observed in prokaryotic and eukaryotic DNA sequences. These are discussed with respect to some of their biological implications. It is suggested that their origins relate to different specific structures of nearest neighbor base pairs. These patterns strongly constrain the DNA sequence. As such, they "explain" to some degree the amino acid codon choice and have direct bearing on questions related to evolution.     

Nearest neighbor relationships of the polypeptides in ubiquinone cytochrome c reductase (complex III).

Ubiquinone cytochrome c reductase (complex III) in detergent dispersion has been cross-linked with two reversible cross-linking agents dithiobissuccinimidylpropionate and dimethyl-3.3'-dithiobispropionimidate and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, polypeptides I and II, II and VI, I and V, and VI and VII were the most prominent subunit pairs seen. With higher levels of reagent, larger aggregates were produced until an aggregate of apparent molecular weight 310 000 was the dominant band on gels. This is the complex III monomer.     

Nearest neighbor effects on carcinogen binding to guanine runs in DNA.

A synthetic DNA fragment was constructed to determine the effect of 5' and 3' neighbors of guanine runs on the binding of chemical carcinogens. Determinations were made on the relative intensity of reactivity between aflatoxin B1 or benzo(a)pyrene and methylnitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with various guanine positions in an endlabeled DNA fragment of known sequence. After reaction, the fragments were depurinated to produce strand breaks to allow Maxam and Gilbert sequencing for guanine positions. Relative reaction intensities were compared densitometrically. 3' neighbors exerted greater influence on carcinogen binding than did 5' neighbors, the influence extended only to the adjacent guanine and depended upon the chemical nature of the carcinogen. In addition, the presence of one carcinogen adduct in the guanine run influenced the formation of a subsequent adduct when the DNA was exposed to a second carcinogen, and this effect also depended on the nature of the second carcinogen. The results suggest that DNA adduct formation in the presence of multiple carcinogens is more complicated than an additive mechanism would suggest.     

Atomic model of the E. coli membrane-bound protein translocation complex SecYEG

The Sec complex forms the core of a conserved machinery transporting proteins across or into membranes. In Escherichia coli SecYEG is active as an oligomer, but the structure predicts that the protein-conducting channel is formed by the monomer. A homology model of the E.coli complex was built using the atomic structure of Methanococcus jannaschii SecYEbeta. Another structure of the membrane-bound dimer was then determined by fitting the homology model to an 8A map of SecYEG determined by electron microscopy. We found that the substrate-binding site of the dimer has opened slightly and the plug domain moved toward the outside. This new position retains the channel in a closed state. These differences partially reflect the movements that have been proposed to occur during channel gating. Further opening of the substrate-binding pocket to bind and release bound substrate and displacement of the plug during secretion, presumably rely on the action of the partner proteins. The contacts arising at the dimer interface in the environment of the lipid bilayer may have activated the assembly.     

Characterization of the cytosolic tuberin-hamartin complex. Tuberin is a cytosolic chaperone for hamartin

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction and dermatological abnormalities. Mutations to either the TSC1 or TSC2 gene are responsible for the disease. The TSC1 gene encodes hamartin, a 130-kDa protein without significant homology to other known mammalian proteins. Analysis of the amino acid sequence of tuberin, the 200-kDa product of the TSC2 gene, identified a region with limited homology to GTPase-activating proteins. Previously, we demonstrated direct binding between tuberin and hamartin. Here we investigate this interaction in more detail. We show that the complex is predominantly cytosolic and may contain additional, as yet uncharacterized components alongside tuberin and hamartin. Furthermore, because oligomerization of the hamartin carboxyl-terminal coiled coil domain was inhibited by the presence of tuberin, we propose that tuberin acts as a chaperone, preventing hamartin self-aggregation.     

Molecular analysis of the cytosolic Dictyostelium gamma-tubulin complex

gamma-Tubulin plays an essential role in microtubule nucleation and organization and occurs, besides its centrosomal localization, in the cytosol, where it forms soluble complexes with other proteins. We investigated the size and composition of gamma-tubulin complexes in Dictyostelium, using a mutant cell line in which the endogenous copy of the gamma-tubulin gene had been replaced by a tagged version. Dictyostelium gamma-tubulin complexes were generally much smaller than the large gamma-tubulin ring complexes found in higher organisms. The stability of the small Dictyostelium gamma-tubulin complexes depended strongly on the purification conditions, with a striking stabilization of the complexes under high salt conditions. Furthermore, we cloned the Dictyostelium homolog of Spc97 and an almost complete sequence of the Dictyostelium homolog of Spc98, which are both components of gamma-tubulin complexes in other organisms. Both proteins localize to the centrosome in Dictyostelium throughout the cell cycle and are also present in a cytosolic pool. We could show that the prevailing small complex present in Dictyostelium consists of DdSpc98 and gamma-tubulin, whereas DdSpc97 does not associate. Dictyostelium is thus the first organism investigated so far where the three proteins do not interact stably in the cytosol.     

Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA

The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.     

Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex

The bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA–SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation.     

Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex.

The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr. Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to [32P]p(dT)16 and 70% of the nonspecific binding energy of the intact protein. However, peptide CN6 does not exhibit the binding specificity of the intact protein. Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.     

Synthetic peptides identify a second periplasmic site for the plug of the SecYEG protein translocation complex

A short helix in the centre of the SecY subunit serves as a ‘plug’ blocking the protein channel. This site must be vacated if the channel is to open and accommodate translocating protein. We have synthesised a peptide mimic of this plug, and show that it binds to E. coli SecYEG, identifying a distinct and peripheral binding site. We propose that during active translocation the plug moves to this second discrete site and chart its position. Deletion of the plug in SecY increases the stoichiometry of the peptide-SecYEG interaction by also exposing the location it occupies in the channel. Binding of the plug peptide to the channel is unaffected by SecA.     

Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.     

Identification of cytosolic Mg2+-dependent soluble inorganic pyrophosphatases in potato and phylogenetic analysis

Using polyclonal antibodies raised against a previously cloned potato Mg2+-dependent soluble inorganic pyrophosphatase (ppa1 gene) [8], a second gene, called ppa2, could be isolated. A single locus homologous to ppa2 was mapped on potato chromosomes, unlinked to the two loci identified for ppa1. From a phylogenetic and structural point of view, the PPA1 and PPA2 polypeptides are more closely related to prokaryotic than to eukaryotic Mg2+-dependent soluble inorganic pyrophosphatases (soluble PPases). Subcellular localization by immunogold electron microscopy, using sections from leaf parenchyma cells, showed that PPA1 and PPA2 are localized to the cytosol. Based on these observations, the likely phylogenetic origin and the physiological significance of the cytosolic soluble pyrophosphatases are discussed.     

Cryo-EM structure of the cytosolic AhR complex

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Trypanosoma brucei: Nearest neighbor analysis on the major variable surface coat glycoprotein—Crosslinking patterns with intact cells

Cleavable Crosslinking reagents were used to study interactions among proteins of the surface coat of Trypanosoma brucei. The proteins were resolved by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When intact cells were treated with dithiobis(succinimidylpropionate), we obtained extensive intermolecular Crosslinking of major variable surface coat glycoprotein (VSCG) molecules. This reagent generated no apparent crosslinks between VSCG and other membrane-associated proteins. Complete conversion to oligomers equal to or greater than octamers occurred within 20 min. When purified VSCG in solution was treated with dithiobis(succimidylpropionate), dimers were found. A complex of Cu²⁺ and 1,10-phenanthroline was used to catalyze air oxidation of adjacent sulfhydryls to disulfide bonds; however, no crosslinking among VSCG molecules nor between VSCG and other proteins was observed. The results presented indicate that VSCG in solution exists predominately in the form of dimers. Whether VSCG in situ also occurred as dimers could not be determined; however, since we observed trimeric and tetrameric forms of VSCG when untreated cells were analyzed, it is likely that weak interactions occur among the protein molecules. These interactions are less stable than the dimer association observed with purified VSCG. Finally, the analysis indicated that VSCGs of this stock of T. brucei, derived from UGANDA/ 60/TREU/164[ETat3], contained at least one intramolecular disulfide bond. We examined T. brucei stocks 427 and EATRO 110 and obtained similar results. Thus, it appears that intramolecular disulfide bonding is a general feature of T. brucei VSCGs.     

Binding of SecA to the SecYEG complex accelerates the rate of nucleotide exchange on SecA

SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex. Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction. Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein. This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction.     

Identification and characterization of a cytosolic protein tyrosine kinase of HeLa cells.

Fractionation of a cytosolic extract of HeLa cells revealed the existence of a highly active protein tyrosine kinase. Chromatographic fractionation of the extract resulted in partial purification of a single enzymatic activity that coeluted with a 94-kDa polypeptide. In vitro phosphorylation of the isolated enzyme showed that p94 was the only polypeptide phosphorylated and only the tyrosine residue(s) was (were) modified. The fractionated enzyme (p94 kinase) also phosphorylated a number of other nonspecific substrates exclusively on tyrosine residues. Unlike other protein tyrosine kinases that have been characterized, p94 kinase is relatively insensitive to inhibition by the isoflavone genistein. Using two different antisera, we provided evidence that the HeLa p94 kinase is most likely the FER gene product, which was previously shown to be expressed in a wide variety of cell types. These results represent the first biochemical characterization of the cellular FER gene product and also provide a basis for studying the biochemistry of tyrosine kinase function in HeLa cells.