Characterization of functionally important sites in the bacteriophage Mu transposase protein

The 663 amino acid Mu transposase protein is absolutely required for Mu DNA transposition. Mutant proteins were constructed in vitro in order to locate regions of transposase that may be important for the catalysis of DNA transposition. Deletions in the A gene, which encodes the transposase, yielded two stable mutant proteins that aid in defining the end-specific DNA-binding domain. Linker insertion mutagenesis at eight sites in the Mu A gene generated two proteins, FF6 and FF14 (resulting from two and four amino acid insertions, respectively, at position 408), which were thermolabile for DNA binding in vitro at 43°C. However, transposition activity in vivo was severely reduced for all mutant proteins at 37°C, except those with insertions at positions 328 and 624. In addition, site-specific mutagenesis was performed to alter tyrosine 414, which is situated in a region that displays amino acid homology to the active sites of a number of nicking/closing enzymes. Tyrosine 414 may reside within an important, yet non-essential, site of transposase, as an aspartate-substituted protein had a drastically reduced frequency of transposition, while the remaining mutants yielded reduced, but substantial, frequencies of Mu transposition in vivo.     

The phage Mu repressor c and IS30 transposase proteins are significantly related

The IS30 transposase exhibits significant amino acid sequence homology to the phage Mu repressor c in the amino- and carboxy-terminal regions of the proteins. The conserved sequences include the proposed Mu repressor DNA binding site, which is also related to the proposed Mu and D108 transposase DNA binding sites. The carboxy-terminal homologies are characterised by two almost complete, and one partial, somewhat diverged amino acid sequence repeats. Only weak homologies to this domain are present in the Mu transposase (Mu A). Nevertheless, a clear link between an insertion sequence and a bacteriophage has been established.     

Cloning and nucleotide sequence of the chlD locus

The nucleotide sequence of a Sau3A1 restriction nuclease fragment that complemented an Escherichia coli chlD::Mu cts mutant strain was determined. DNA and deduced amino acid sequence analysis revealed two open reading frames (ORFs) that potentially codes for proteins with amino acid sequence homology with binding protein-dependent transport systems. One of the ORFs showed a sequence that encoded a protein with properties that were characteristic of a hydrophobic inner membrane protein. The other ORF, which was responsible for complementing a chlD mutant, encoded a protein with conserved sequences in nucleotide-binding proteins and hydrophilic inner membrane proteins in active transport systems. A proposal that the chlD locus is the molybdate transport operon is discussed in terms of the chlD phenotype.     

A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions.

The functional configuration of Mu transposase (A protein) is its tetrameric form. We present here a model for the organization of a functional Mu A tetramer. Within the tetramer, assembly of each of the two active sites for Mu end cleavage requires amino acid contributions from the central and C-terminal domains (domains II and III respectively) of at least two Mu A monomers in a trans configuration. The Mu enhancer is likely to function in this assembly process by specifying the two monomers that provide their C-terminal domains for strand cleavage. The Mu B protein is not required in this step. Each of the two active sites for the strand transfer reaction is also organized by domain sharing (but in the reverse mode) between Mu A monomers; i.e. a donor of domain II (also the recipient of domain III) during cleavage is a recipient of domain II (and the donor of domain III) during strand transfer. The function of the Mu B protein (which is required at the strand transfer step) and that of the enhancer element may be analogous in that their interactions with Mu A (domain III and domain I alpha respectively) promote conformations of Mu A conducive to strand cleavage or strand transfer.     

Cloning and expression in Escherichia coli of a gene,hup, encoding the histone-like protein HU of Bifidobacterium longum

A genomic DNA library of Bifidobacterium longum ATCC15707 was transfected into an Escherichia coli strain deficient in both HU and IHF, the growth of which is cold-sensitive because of the deficiency in these proteins. Cold-resistant colonies were selected and the DNA was cloned and sequenced. A polypeptide consisted of 93 amino acids, a predicted molecular mass of 9983 Da with an isoelectric point of 10.35, was deduced from an orf in the middle of the DNA fragment. The amino acid sequence was highly similar to HU family proteins, and 26 aas of N terminal was identical to a histone-like protein, HBI, a HU family protein of B. longum. Incapabilities of Mu phage propagation in an E. coli mutant deficient in HU or IHF could be suppressed by DNA bearing this orf. These results showed that the orf is a gene hup encoding HBI, a histone-like protein HU of B. longum.     

Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.     

A subsequence-specific DNA-binding domain resides in the 13 kDa amino terminus of the bacteriophage Mu transposase protein

We have previously reported that the 13 kDa amino terminus of the 70 kDa bacteriophage D108 transposase protein (A gene product) contains a two-component, sequence-specific DNA-binding domain which specifically binds to the related bacteriophage Mu's right end (attR) in vitro. To extend these studies, we examined the ability of the 13 kDa amino terminus of the Mu transposase protein to bind specifically to Mu attR in crude extracts. Here we report that the Mu transposase protein also contains a Mu attR specific DNA-binding domain, located in a putative alpha-helix-turn-alpha-helix region, in the amino terminal 13 kDa portion of the 70 kDa transposase protein as part of a 23 kDa fusion protein with beta-lactamase. We purified for this attR-specific DNA-binding activity and ultimately obtained a single polypeptide of the predicted molecular weight for the A'--'bla fusion protein. We found that the pure protein bound to the Mu attR site in a different manner compared with the entire Mu transposase protein as determined by DNase I-footprinting. Our results may suggest the presence of a potential primordial DNA-binding site (5'-PuCGAAA-3') located several times within attR, at the ends of Mu and D108 DNA, and at the extremities of other prokaryotic class II elements that catalyze 5 base pair duplications at the site of element insertion. The dissection of the functional domains of the related phage Mu and D108 transposase proteins will provide clues to the mechanisms and evolution of DNA transposition as a mode of mobile genetic element propagation.     

Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA.

Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.     

The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review)

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.     

Amino acid sequence of a peptide from keratan sulfate II-core protein linkage regions

Keratan sulfate II was prepared from the proteolytic digest of pig nucleus pulposus proteoglycan. The polysaccharide chains containing the fragment peptides of the core protein at their reducing terminal were subjected to anhydrous HF-solvolysis reaction and one of the glycopeptides from the keratan sulfate II-core protein linkage regions was isolated. The amino acid sequence of the peptide was deduced to be Ala-Pro-Ser-Pro-Gly, which is different from those reported for the attachment sites of chondroitin sulfate on core proteins from various sources. The results provided the first solid amino acid sequence for the keratan sulfate II-core protein linkage regions and suggested that the amino acid sequence of the core protein might determine the distribution of chondroitin sulfates and keratan sulfates along the core protein of the proteoglycan molecule.     

Instability of transposase activity: Evidence from bacteriophage Mu DNA replication

Transposition of genetic elements involves coupled replication and integration events catalyzed in part by a class of proteins called transposases. We have asked whether the transposase activity of bacteriophage Mu (the Mu A protein) is stable and capable of catalyzing multiple rounds of coupled replication/integration, or whether its continued synthesis is required to maintain Mu DNA replication. Inhibition of protein synthesis during the lytic cycle with chloramphenicol inhibited Mu DNA synthesis with a half-life of approximately 3 min, demonstrating a need for continued protein synthesis to maintain Mu DNA replication. Synthesis of specific Mu-encoded proteins was inhibited by infecting a host carrying a temperature-sensitive suppressor, at permissive temperature, with Mu amber phages, then shifting to nonpermissive temperature. When Aam phages were used, Mu DNA replication was inhibited with kinetics essentially identical to those with chloramphenicol addition; hence, it is likely that continued synthesis of the Mu A protein is required to maintain Mu DNA replication. The data suggest that the activity of the Mu A protein is unstable, and raise the possibility that the Mu A protein and other transposases may be used stoichiometrically rather than catalytically.     

Cytosolic rat liver glutathione transferase 4-4. Primary structure of the protein reveals extensive differences between homologous glutathione transferases of classes alpha and mu

The primary structure of the class Mu glutathione transferase 4-4 from rat liver was determined. The structural data characterize a class Mu protein within an enzyme family for which three classes have been distinguished (Alpha, Mu, Pi). The structure was determined by analysis of peptides obtained after treatment with trypsin. Glu-specific protease and CNBr. The protein is composed of two identical subunits, each with 217 amino acid residues. No evidence for microheterogeneity or for the presence of modified residues was encountered. The primary structure was found to be strictly homologous with corresponding parts in known regions of other class Mu enzymes of rat, mouse, human and bovine origin. Relationships to the cytosolic enzyme of other classes (Pi and Alpha) are considerably more distant. A comparison with the entire chain of the class Alpha subunit 1 from rat liver was carried out by three methods, alignment of amino acid sequences, correlation of hydrophilicity plots and predictions of secondary structures. All methods reveal weak similarities but also large differences. The overall positional identity is only 26%. Combined, the results establish the first complete class Mu structure, show distant inter-class relationships, and relate subunit 4 (class Mu) and subunit 1 (class Alpha) in a family of enzymes rather than in a group of isoenzymes.     

Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu

The construction is described of a plasmid (pL-ner) which directs the high-level production of the bacteriophage Mu Ner protein in Escherichia coli. The protein, recovered in the soluble cellular fraction, was susceptible to in vivo proteolytic processing, in many host strains, but not in E. coli B, a natural lon- prototroph. A simple purification method is described which takes advantage of the basic nature of the protein. The purified protein was shown to be physically and chemically homogeneous and to have an amino acid sequence identical to that predicted for the authentic protein. The protein was also shown to have in vitro biological activity, as measured by specific binding to a DNA fragment containing the consensus Ner-binding sequence, and in vivo biological activity as the protein produced by the pL-ner plasmid allowed lysogenic-like maintenance of a Mu prophage c mutant unable to synthesise a functional Mu repressor.     

Protein fatty acylation: a novel mechanism for association of proteins with membranes and its role in transmembrane regulatory pathways

A wide range of proteins of cellular and viral origin have been shown to be modified covalently by long-chain fatty acids. Recent studies have revealed at least two distinct types of protein fatty acylation which involve different fatty acyltransferases. The abundant fatty acid, palmitate, is incorporated post-translationally through a thiol ester linkage into a variety of cell surface glycoproteins and non-glycosylated intracellular proteins. In contrast, the rare fatty acid, myristate, is incorporated co-translationally through an amide linkage into numerous intracellular proteins. Identification of proteins that contain covalent fatty acids has revealed that this modification is common to a broad array of proteins that play important roles in transmembrane regulatory pathways. For many of these proteins, the fatty acid moiety appears to play an important role in directing the polypeptide to the appropriate membrane and in mediating protein-protein interactions within the membrane. This review will summarize recent studies that define different pathways for protein fatty acylation and will consider the potential functions for this unique covalent modification of proteins.