The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.     

Reversion of temperature-sensitive mutation by inhibition of proteasome-mediated degradation of mutated D123 protein.

A temperature-sensitive cell-cycle mutant of the 3Y1 rat fibroblast cell line, 3Y1tsD123 has in the D123 gene coding region a point mutation which causes instability of the D123 protein. Temperature-sensitive G1 arrest of the mutant is caused by increased degradation of the D123 protein at restrictive temperature. In this study we found that the selective proteasome inhibitors lactacystin and MG132 inhibited degradation of the mutated D123 protein in cell lines overexpressing the mutated D123 protein, followed by accumulation of a modified form (increased molecular weight other than by ubiquitination) of the D123 protein. Although a temperature-resistant revertant of the mutant had no further mutation in the D123 gene coding region, the modification of the mutated D123 protein was inhibited and the mutated D123 protein was rendered stable. The modification was also inhibited in the hybrid cell lines between the revertant and the cell line overexpressing the mutated D123 protein. These facts imply that the mutated D123 protein receives unidentified modification before degradation in the proteasome, and that the revertant expresses a gene inhibiting this modification.     

Enhanced Mutability Associated with a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.     

Fully metallated S134N Cu,Zn-superoxide dismutase displays abnormal mobility and intermolecular contacts in solution

S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metal-deficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. II. Biochemical Evidence for Genetic Exchange

The genome of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol. 24:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the ts phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R. HaeIII cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the tsD lesion), is missing in the DNAs of the parental tsD202 virus and of three independent revertants of tsD202. In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued D202 populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R. Hin(II + III) cleavage patterns of SV40 strain 777 DNA and tsD202 DNA revealed differences in the electrophoretic mobilities of Hin fragments A, B, and F. However, the corresponding Hin fragments from all four rescued D202 genomes were identical in their mobilities to those of tsD202 DNA, indicating that these regions of the rescued D202 genome are characteristic of the tsD202 parent. We conclude, therefore, that the genome of the rescued D202 virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous tsD202 mutant.     

Albumin Hawkes Bay; a low level variant caused by loss of a sulphydryl group at position 177

A slow migrating minor albumin component, representing 5% of total circulating albumin, was detected by routine serum protein electrophoresis and immunofixation. After treatment with 5 mM dithiothreitol the abnormal component was found to migrate normally suggesting the attachment of some component to the free thiol at position 34. However, purification and analysis by SDS-PAGE showed that the abnormal component had a slightly lower apparent molecular weight than normal albumin. Limited tryptic cleavage indicated the abnormal site to be in the N-terminal third of the molecule. HPLC analysis of tryptic peptides from this domain showed the presence of a new peptide of sequence Ala-Ala-Phe-Leu-Leu-Pro-Lys, indicating either a point mutation of 177 Cys → Phe or the deletion of residues 166–177. DNA sequencing of PCR-amplified DNA confirmed the former Cys → Phe substitution by indicating a point mutation of C to A at nucleotide position 5185. It appears that the aberrant electrophoretic mobility of the variant might be due to a gross conformational change associated with the formation of a new disulphide bond between Cys-168 and Cys-124.     

Alteration of ribosomal protein S17 by mutation linked to neamine resistance in Escherichia coli: II. Localization of the amino acid replacement in protein S17 from a neaA mutant

A mutant of Escherichia coli strain K12S, nea^R301, resistant to the antibiotic neamine was found to have an altered 30 S ribosomal protein S17. The modification involves a change in the electrophoretic mobility of this protein. S17 proteins wore purified from the mutant and the parental strain, respectively, and the amino acid compositions of all tryptic peptides were compared. The results show that the mutational alteration involves a replacement of histidine by proline in peptide T8 from mutant nea^R301. The amino acid replacement is located at position 30 of the S17 protein chain. We conclude, therefore, that the mutation nea^R301 affects the structural gene for protein S17 (rps Q).     

Phenylalanine transport in Aspergillus nidulans: demonstration of role of phenylalanine binding proteins

Crude shock proteins extracted by two stage osmotic shock were further purified by affinity chromatography to obtain ligand (phenylalanine) specific binding protein (phebip) a component of phenylalanine (phe) transport system from wild type and a phe transport mutant fpaD11 of Aspergillus nidulans. A new eluent 0.1 M Tris-HCl containing 1.5 N NaCl and 0.5 N Na2CO3, pH 8 was used during the investigation. The elution profile of mutant phebip exhibited one simple and two compound peaks instead of three simple ones as exhibited by the wild type phebip. SDS-PAGE profile of mutant phebip showed faster electrophoretic mobility than that of wild type one. It is therefore evident that the mutant phebip has reduced molecular mass (M(r)) due to deletion of a segment that somehow has bearing on the binding capacity of the active site of phebip. The resultant erosion in the binding capacity of the mutant phebip is in turn responsible for its incapability to stimulate transport of ligand across the plasma membrane.     

Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase

The CYR3 mutant of yeast, Saccharomyces cerevisiae, partially accumulated unbudded cells and required cAMP for the best growth at 35 degrees C. The CYR3 mutation was partially dominant over the wild type counterpart and suppressed by the bcy1 mutation which is responsible for the deficiency of the regulatory subunit of cAMP-dependent protein kinase. The molecular weights of cAMP-dependent protein kinase and its catalytic and regulatory subunits were 160,000, 30,000, and 50,000, respectively. No significant differences in the molecular weights of cAMP-dependent protein kinase and the subunits were found between the wild type and CYR3 mutant strains. However, the cAMP-dependent protein kinase activity of CYR3 cells showed significantly higher Ka values for activation by cAMP at 35 degrees C than those of wild type and a clear difference in the electrophoretic mobility of the regulatory subunit was found between the wild type and CYR3 enzymes. The CYR3 mutation was suppressed by the IAC mutation which caused the production of a significantly high level of cAMP. The results indicate that the CYR3 phenotype was produced by a structural mutation in the CYR3 gene coding for the regulatory subunit of cAMP-dependent protein kinase in yeast.     

A mutation in the C-terminal putative Zn2+ finger motif of UL52 severely affects the biochemical activities of the HSV-1 helicase-primase subcomplex

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex that is composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins retains all the enzymatic activities exhibited by the holoenzyme in vitro. The UL52 protein contains a putative zinc finger at its C terminus which is highly conserved among both prokaryotic and eukaryotic primases. We constructed a mutation in which two highly conserved cysteine residues in the zinc finger motif were replaced with alanine residues. A UL52 expression plasmid containing the mutation in the zinc finger region is unable to support the growth of a UL52 mutant virus in a transient complementation assay. Wild type and mutant UL5.UL52 subcomplexes were purified from insect cells infected with recombinant baculoviruses. Surprisingly, the mutant protein was severely affected in all biochemical activities tested; no helicase or primase activities could be detected, and the mutant protein retains only about 9% of wild type levels of single-stranded DNA-dependent ATPase activity. Gel mobility shift assays showed that DNA binding is severely affected as well; the mutant subcomplex only retains approximately 8% of wild type levels of binding to a forked substrate. On the other hand, the mutant protein retains its ability to interact with UL5 as indicated by copurification and with UL8 as indicated by a supershifted band in the gel mobility shift assay. In addition, the ability of individual subunits to bind single-stranded DNA was examined by photo cross-linking. In the wild type UL5.UL52 subcomplex, both subunits are able to bind an 18-mer of oligo(dT). The mutant subcomplex was severely compromised in the ability of both UL5 and UL52 to bind the oligonucleotide; total cross-linking was only 2% of wild type levels. These results are consistent with the proposal that the putative zinc binding motif of UL52 is required not only for binding of the UL52 subunit to DNA and for primase activity but also for optimal binding of UL5 to DNA and for the subsequent ATPase and helicase activities.     

Analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted RNA recombination.

The Alb4 mutant of the coronavirus mouse hepatitis virus (MHV) is both temperature sensitive and thermolabile owing to a deletion in the gene encoding its nucleocapsid (N) protein. The deletion removes 29 amino acids that constitute a putative spacer region preceding the carboxyl-terminal domain of the protein. As a step toward understanding the structure and function of the MHV N protein, we isolated multiple independent revertants of Alb4 that totally or partially regained the ability to form large (wild-type-sized) plaques at the nonpermissive temperature. The N proteins of these revertant viruses concomitantly regained the ability to bind to RNA in vitro at a temperature that was restrictive for RNA binding by Alb4 N protein. Sequence analysis of the N genes of the revertants revealed that each contained a single second-site point mutation that compensated for the effects of the deletion. All reverting mutations were clustered within a stretch of 40 amino acids centered some 80 residues on the amino side of the Alb4 deletion, within a domain to which the RNA-binding activity of N had been previously mapped. By means of a targeted RNA recombination method that we have recently developed, two of the reverting mutations were introduced into a wild-type MHV genomic background. The resulting recombinants were stable and showed no gross phenotypic differences from the wild type. A detailed analysis of one, however, revealed that it was at a selective disadvantage with respect to the wild type.     

Ribosomal proteins

Summary Ribosomal proteins fromE. coli mutant N421 which is a spontaneous revertant from streptomycin dependence to independence have been compared to those of the wild type by two-dimensional polyacrylamide gel electrophoresis and by four immunochemical methods. The only detectable difference is a change in protein S5. This finding suggests that reversion from streptomycin dependence to independence can be caused not only by a mutation in protein S4, as described earlier, but also in protein S5.     

Biochemical analysis of att-defective mutants of the phage lambda site-specific recombination system

Three mutations previously mapped to the common core region of the bacteriophage lambda att site have been sequenced. All were found to be due to the deletion of a T residue from a string of six T residues within the 15 base-pair core, the region of homology between the recombining sites. As judged by DNAase I protection experiments, binding of the Int protein is the same in the mutant and wild-type core sites. However, a difference in the Int binding to mutant cores is observed when the small neocarzinostatin molecule is used as a nuclease probe. The differences between mutant and wild type lead to the suggestion that Int is interacting with sequences at the core-arm junctions. Accordingly, the mutants are proposed to be defective in the spacing of Int monomers bound at two recognition sequences spanning the core-arm junctions. The anomalous electrophoretic mobility of wild-type att fragments and, more specifically, the effect of the single base core deletion on electrophoretic mobility are discussed in the text and in the Appendix. The mutant att²501, defective in both att and int functions, was sequenced and found to be a 335 base-pair deletion removing the coding region for 25 amino acids from the carboxy-terminal end of Int, as well as the entire att site. The postulated origin of the 501 mutation is also consistent with the model of two juxtaposed Int recognition sites.     

The Molecular Basis of Brown, an Old Mouse Mutation, and of an Induced Revertant to Wild Type

The murine b locus encodes the tyrosinase related protein, TRP-1, a putative membrane-bound, copper-containing enzyme having about 40% amino acid identity with tyrosinase. The protein is essential for production of black rather than brown hair pigment. We show that skin of mutant brown mice contains the same amount of TRP-1 mRNA as wild type. On sequencing the coding region of the mutant mRNA we find four nucleotide differences from the wild-type (Black) sequence. Two of these differences result in different amino acid residues encoded by the brown allele. By sequencing the TRP-1 gene from a mouse in which a reversion from brown to Black has been induced by ethylnitrosourea we are able to show that only one of these amino acid changes, which substitutes a tyrosine for a conserved cysteine, is the cause of the brown phenotype. This mutation is adjacent to another cysteine at which, in the analogous position in tyrosinase a mutation results in the albino phenotype. The sequence of the revertant is the first report of DNA sequence of an ethylnitrosourea-induced genetic change in mouse.     

Energetics of proton transfer pathways in reaction centers from Rhodobacter sphaeroides. The Glu-H173 activated mutants

Electron transfer between the primary and secondary quinones (Q(A), Q(B)) in the bacterial photosynthetic reaction center (bRC) is coupled with proton uptake at Q(B). The protons are conducted from the cytoplasmic side, probably with the participation of two water channels. Mutations of titratable residues like Asp-L213 to Asn (inhibited mutant) or the double mutant Glu-L212 to Ala/Asp-L213 to Ala inhibit these electron transfer-coupled proton uptake events. The inhibition of the proton transfer (PT) process in the single mutant can be restored by a second mutation of Arg-M233 to Cys or Arg-H177 to His (revertant mutant). These revertant mutants shed light on the location of the main proton transfer pathway of wild type bRC. In contrast to the wild type and inhibited mutant bRC, the revertant mutant bRC showed notable proton uptake at Glu-H173 upon formation of the Q(B)- state. In all of these mutants, the pK(a) of Asp-M17 decreased by 1.4-2.4 units with respect to the wild type bRC, whereas a significant pK(a) upshift of up to 5.8 units was observed at Glu-H122, Asp-H170, Glu-H173, and Glu-H230 in the revertant mutants. These residues belonging to the main PT pathway are arranged along water channel P1 localized mainly in subunit H. bRC possesses subunit H, which has no counterpart in photosystem II. Thus, bRC may possess alternative PT pathways involving water channels in subunit H, which becomes active in case the main PT pathway is blocked.     

A single amino acid change in subunit 6 of the yeast mitochondrial ATPase suppresses a null mutation in ATP10

In an earlier study, the ATP10 gene of Saccharomyces cerevisiae was shown to code for an inner membrane protein required for assembly of the F(0) sector of the mitochondrial ATPase complex (Ackerman, S., and Tzagoloff, A. (1990) J. Biol. Chem. 265, 9952-9959). To gain additional insights into the function of Atp10p, we have analyzed a revertant of an atp10 null mutant that displays partial recovery of oligomycin-sensitive ATPase and of respiratory competence. The suppressor mutation in the revertant has been mapped to the OLI2 locus in mitochondrial DNA and shown to be a single base change in the C-terminal coding region of the gene. The mutation results in the substitution of a valine for an alanine at residue 249 of subunit 6 of the ATPase. The ability of the subunit 6 mutation to compensate for the absence of Atp10p implies a functional interaction between the two proteins. Such an interaction is consistent with evidence indicating that the C-terminal region with the site of the mutation and the extramembrane domain of Atp10p are both on the matrix side of the inner membrane. Subunit 6 has been purified from the parental wild type strain, from the atp10 null mutant, and from the revertant. The N-terminal sequences of the three proteins indicated that they all start at Ser(11), the normal processing site of the subunit 6 precursor. Mass spectral analysis of the wild type and mutants subunit 6 failed to reveal any substantive difference of the wild type and mutant proteins when the mass of the latter was corrected for Ala --> Val mutation. These data argue against a role of Atp10p in post-translational modification of subunit 6. Although post-translational modification of another ATPase subunit interacting with subunit 6 cannot be excluded, a more likely function for Atp10p is that it acts as a subunit 6 chaperone during F(0) assembly.     

Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.     

Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage

EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.