High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M.EcoR124.

We have cloned the genes coding for the two subunits (HsdM and HsdS) of the type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119. These subunits have been synthesized together as an intact MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active MTase. We have partially purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.     

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.     

The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity.

Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.     

Purification and properties of a ribosomal protein methylase from Eschericha coli Q13.

Ribosomal protein methylase has been purified from Escherichia coli strain Q13 using methyl-deficient 50S subunits as substrates. The purified enzyme (or enzyme complex) which is devoid of rRNA methylating activity is quite stable and has a pH optimum around 8.0. The Km for S-adenosyl-L-methionine is 3.2 muM. The molecular weight of the enzyme is 3.1 X 10(4); minor methylating activity was also detected for protein peaks with molecular weights of 1.7 X 10(4) and 5.6 X 10(4). Protein L11 is the major protein methylated by the purified enzyme. Product analysis revealed the presence of N epislon-trimethyllysine, a methylated neutral amino acid(s) previously observed in protein L11 and N epislon-monomethyllysine. Free ribosomal proteins were much better substrates for the methylation, indicating that methylation of 50S ribosomal proteins can occur before the complete assembly of the 50S ribosomal subunit.     

Enzymatic methyl esterification of Escherichia coli ribosomal proteins.

Enzymatic methyl ester formation in Escherichia coli ribosomal proteins was observed. Alkali lability of the methylated proteins and derivatization of the methyl groups as methyl esters of 3,5-dinitrobenzoate indicate the presence of protein methyl esters. The esterification reaction occurs predominantly on the 30S ribosomal subunit, with protein S3 as the major esterified protein. When the purified 30S subunit was used as the methyl acceptor, protein S9 was also found to be esterified. The enzyme responsible for the esterification of free carboxyl groups in proteins, protein methylase II (S-adenosyl-L-methionine:protein carboxyl methyltransferase, EC 2.1.1.24), was identified in E. coli Q13. This enzyme is extremely unstable when compared with that from mammalian origin. By molecular sieve chromatography, E. coli protein methylase II showed multiple peaks, with a major broad peak around 120,000 daltons and several minor peaks in the lower-molecular-weight region. Rechromatography of the major enzyme peak showed activities in several fractions that are much lower in molecular weight. The substrate specificity of the E. coli enzyme is similar to that of the mammalian enzyme. The Km value for S-adenosyl-L-methionine is 1.96 X 10(-6) M, and S-adenosyl-L-homocysteine was found to be a competitive inhibitor, with a Ki value of 1.75 X 10(-6) M.     

The Doc1 subunit is a processivity factor for the anaphase-promoting complex

Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APC(doc1 Delta)) indicates that Doc1 is required for processivity. The specific molecular defect in APC(doc1 Delta) is identified by a large increase in apparent K(M) for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APC(doc1 Delta) fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate-enzyme affinity.     

Large-scale purification and characterization of the five subunits of F1-ATPase from pig heart mitochondria.

A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits.     

Oligomerization and ligand-binding properties of the ectodomain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluRD.

The extracellular part of ionotropic glutamate receptor (iGluR) subunits can be divided into a conserved two-lobed ligand-binding domain ("S1S2") and an N-terminal approximately 400-residue segment of unknown function ("X domain") which shows high sequence variation among subunits. To investigate the structure and properties of the N-terminal domain, we have now produced affinity-tagged recombinant fragments which represent the X domain of the GluRD subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptors either alone or covalently linked to the ligand-binding domain ("XS1S2"). These fragments were expressed in insect cells as secreted soluble proteins and were recognized by a conformation-specific anti-GluRD monoclonal antibody. A hydrodynamic analysis of the purified fragments revealed them to be dimers, in contrast to the S1S2 ligand-binding domain which is monomeric. The X domain did not bind radiolabeled AMPA or glutamate nor did its presence affect the ligand binding properties of the S1S2 domain. Our findings demonstrate that the N-terminal domain of AMPA receptor can be expressed as a soluble polypeptide and suggest that subunit interactions in iGluR may involve the extracellular domains.     

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

Proteins encoded by the DpnII restriction gene cassette. Two methylases and an endonuclease

Proteins encoded by three genes in the DpnII restriction enzyme cassette of Streptococcus pneumoniae were purified and characterized. Large amounts of the proteins were produced by subcloning the cassette in an Escherichia coli expression system. All three proteins appear to be dimers composed of identical polypeptide subunits. One is the DpnII endonuclease, and the other two are DNA adenine methylase active at 5' GATC 3' sites. Inactivation of enzyme activity by insertions into the genes and comparison of the DNA sequence with the amino-terminal sequence of amino acid residues in the proteins demonstrated the following correspondence between genes and enzymes. The promoter-proximal gene in the operon, dpnM, encodes a 33 X 10(3) Mr polypeptide that gives rise to a potent DNA methylase. The next gene, dpnA, encodes the 31 x 10(3) Mr polypeptide of a weaker and less-specific methylase. The third gene, dpnB, encodes the 34 x 10(3) Mr polypeptide of the endonuclease. Although the endonuclease polypeptide is initiated from an ordinary ribosome-binding site, each of the methylase polypeptide begins at an atypical site with a consensus sequence entirely different from that of Shine & Dalgarno. This presumptive novel ribosome-binding site is well recognized in both S. pneumoniae and E. coli.     

Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell

To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Paramecium Has Two Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase, One Unique to Cilia

ABSTRACT. The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R₄₄) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R₄₄ were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R₄₈) weakly recognized by one of several monoclonal antibodies against R₄₄, but not recognized by an anti-R₄₄ polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R₄₈ and a 70-kDa form containing C and R₄₄. In the large enzyme, R₄₈ was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R₄₄ The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R₄₈ were retained by the column and eluted with 1 M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R₄₄, whereas R₄₈ is the R subunit of the large enzyme.     

The subunit structure and dynamics of the 20S proteasome in chicken skeletal muscle

We have succeeded in purifying the 20S core proteasome particle from less than 1 g of skeletal muscle in a rapid process involving two chromatographic steps. The individual subunits were readily resolved by two-dimensional PAGE, and the identities of each of the 14 subunits were assigned by a combination of peptide mass fingerprinting and MS/MS/de novo sequencing. To assess the dynamics of proteasome biogenesis, chicks were fed a diet containing stable isotope-labeled valine, and the rate of incorporation of label into valine-containing peptides derived from each subunit was assessed by mass spectrometric analysis after two-dimensional separation. Peptides containing multiple valine residues from the 20S proteasome and other soluble muscle proteins were analyzed to yield the relative isotope abundance of the precursor pool, a piece of information that is essential for calculation of turnover parameters. The rates of synthesis of each subunit are rather similar, although there is evidence for high turnover subunits in both the alpha (nonproteolytic) and beta (proteolytic) rings. The variability in synthesis rate for the different subunits is consistent with a model in which some subunits are produced in excess, whereas others may be the rate-limiting factor in the concentration of 20S subunits in the cell. The ability to measure turnover rates of proteins on a proteome-wide scale in protein assemblies and in a complex organism provides a new dimension to the understanding of the dynamic proteome.     

Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.     

Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR124I.

The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)(2) is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)(2) complex is able to bind to both the EcoR124I DNA recognition sequence GAAN(6)RTCG and a symmetrical DNA sequence GAAN(7)TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)(2) -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3' and 5' sides of the recognition sequence bound by the (S3/HsdM)(2) complex.     

Removal of a frameshift between the hsdM and hsdS genes of the EcoKI Type IA DNA restriction and modification system produces a new type of system and links the different families of Type I systems

The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence.     

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.     

Comparative proteome analysis of changes in the 26S proteasome during oocyte maturation in goldfish

Proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. An alteration of proteasome function may be important for the regulation of the meiotic cell cycle. To study the change at the subunit level of the 26S proteasome during meiotic maturation, we purified 26S proteasomes from immature and mature oocytes of goldfish. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. For differential analysis, whole spots of the 26S proteasome from goldfish oocytes were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database analysis. Four spots that were different (only detected in mature oocyte 265 proteasomes and not in immature ones) and four protein spots that were up- or down-regulated were identified unambiguously. The mature-specific spots were not 26S proteasome components but rather their interacting proteins, and were identified as chaperonin-containing TCP-1 subunits and myosin light chain. Minor spots of three subunits of the 20S core particle and one of the 19S regulatory particle showed meiotic cell cycle-dependent changes. These results demonstrate that modifications of proteasomal subunits and cell cycle phase-dependent interactions of proteins with proteasomes occur during oocyte maturation in goldfish.     

The effects of triethyltin bromide on red cell and brain cyclic AMP-dependent protein kinases

Triethyltin bromide activates the cyclic AMP-dependent protein kinases of human red cell membranes and of bovine brain. Additions of 25-500 microM triethyltin to red cell ghosts resulted in enhanced phosphorylation of ghost proteins. When added to partially purified cyclic AMP-dependent protein kinases from red cell ghosts or bovine brain, stimulation of the phosphorylation of calf thymus histone was observed. The enhancement of kinase activity was due to release of catalytic subunits from the intact protein kinase. Brief exposure of the partially purified enzymes to triethyltin, followed by DE52 chromatography, resulted in elution profiles for regulatory and catalytic subunits that were similar to the profile resulting after cyclic AMP activation. Triethyltin interacts with both regulatory and catalytic subunits. When it was added to the partially purified cyclic AMP-dependent protein kinases from human red cell ghosts or bovine brain, noncompetitive inhibition of cyclic AMP binding to the regulatory subunit of the enzyme was observed. It interacted with the catalytic subunit to produce slow inhibition of catalytic activity. The inhibition was non-competitive with respect to both histone and ATP. When intact red cells were subjected to brief exposure with triethyltin, enhanced phosphorylation of certain membrane proteins occurred, suggesting that the activation of the cyclic AMP protein kinases by triethyltin may be physiologically significant.