Lawrence Kahan

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran·GTP. Export of 60S subunits is coupled with that of the nucleo‐ cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM‐1‐dependent leucine‐rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild‐type and export‐defective mutant hNMD3 proteins bind to newly made nuclear 60S pre‐export particles at a late step of subunit maturation. The export‐defective hNMD3, but not the wild‐type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild‐type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1–Ran·GTP‐mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.

Ribosomal proteins S5, S11, S13 and S19 localized by ekectron microscopy of antibody-labeled subunits

Specific antibodies prepared against purified Escherichia coli small subunit proteins were used to map protein locations by electron microscopy of bound antibody in negatively contrasted small subunit preparations. By identifying and interpreting characteristic views as projections of a single structure, the protein distributions on the surface of the small subunit have been mapped in three dimensions. Antibodies against ribosomal proteins S5, S11 and S13 bind to single regions of the ribosome surface. In contrast, anti-S19 immunoglobulin G binds to two separated regions of the ribosome surface, suggesting that S19 has an elongated conformation in situ. Proteins S13 and S19 are located on the smaller portion of elongated profiles, while S5 and S11 are located on the larger portion of the subunit profile, near the partition traversing the subunit image. The locations of exposed regions of S11, S13 and S19, in combination with the crosslinking results of others, suggest a binding site for initiation factor IF3 on a region of the 30 S surface, comprising the platform, the cleft, part of the partition, and part of the upper “one third” of the subunit.

Ribosomal proteins S3, S6, S8 and S10 of Escherichia coli localized on the external surface of the small subunit by immune electron microscopy☆

The three-dimensional locations of Escherichia coli ribosomal proteins S3, 86, S8 and S10 on the surface of the small subunit were determined by immune electron microscopy. All four proteins are located on the “external surface” of the small subunit; i.e. on the side of the subunit in contact with the cytosol in the 70 S ribosome. Proteins S3, S6, S8 and S10 map at single sites, although the S3 site is extended approximately 40A along the long axis of the subunit. S8 is located near the base of the cleft separating the platform from the upper one-third or head; protein S10 is located in the head, near the site previously mapped for S14; S3 extends from the level of the constriction to near the top of the head in the vicinity of S10; and S6 is located on the platform of the small subunit near the site previously mapped for S11. The locations of these proteins correlate well with other information on their spatial relationships obtained from assembly interactions, neutron diffraction, crosslinking and protein associations.

Localization of Escherichia coli Ribosomal Proteins S4 and S14 by Electron Microscopy of Antibody-Labeled Subunits

Binding sites for antibodies specific for proteins S4 and S14 of the small subunit of E. coli ribosomes have been mapped on the surface of the subunit by electron microscopy. Antibody binding to reconstituted subunits was shown to depend specifically on the presence of E. coli S4 and S14. Anti-S14 IgG was found to bind to a limited region of the ribosome surface. In contrast anti-S4 IgG was found to bind to three separated regions of the ribosome surface, suggesting S4 has an elongated conformation in situ.

Effects of aminoethylation of cysteine residues on the structural and functional activities of the Escherichia coli 30 S ribosomal proteins.

Abstract The purified 30 S ribosomal proteins from Escherichia coli strain Q13 were chemically modified by reaction with ethyleneimine, specifically converting cysteine residues to S-2-aminoethylcysteine residues. Proteins S1, S2, S4, S8, S11, S12, S13, S14, S17, S18 and S21 were found to contain aminoethylcysteine residues after modification, whereas proteins S3, S5, S6, S7, S9, S10, S15, S16, S19 and S20 did not. Aminoethylated proteins S4, S13, S17 and S18 were active in the reconstitution of 30 S ribosomes and did not have altered functional activities in poly(U)-dependent polyphenylalanine synthesis, R17-dependent protein synthesis, fMet-tRNA binding and Phe-tRNA binding. Aminoethylated proteins S2, S11, S12, S14 and S21 were not active in the reconstitution of complete 30 S ribosomes, either because the aminoethylated protein did not bind stably to the ribosome (S2, S11, S12 and S21) or because the aminoethylated protein did not stabilize the binding of other ribosomal proteins (S14). The functional activities of 30 S ribosomes reconstituted from a mixture of proteins containing one sensitive aminoethylated protein (S2, S11, S12, S14 or S21) were similar to ribosomes reconstituted from mixtures lacking that protein. These results imply that the sulfhydryl groups of the proteins S4, S13, S17 and S18 are not necessary for the structural or functional activities of these proteins, and that aminoethylation of the sulfhydryl groups of S2, S11, S12, S14 and S21 forms either a kinetic or thermodynamic barrier to the assembly of active 30 S ribosomes in vitro.

Production and partial characterization of monoclonal antibodies to Bothrops asper (terciopelo) myotoxin

Seven murine monoclonal antibodies against Bothrops asper myotoxin were produced and partially characterized. They revealed the presence of at least four cross-reacting basic components in crude venom, with a common subunit mol. wt of 16,000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, but slight differences in charge. These myotoxin-related components might be isoforms of the toxin. By Western blotting and enzyme-immunoassay binding techniques, differences in the reactivities with basic venom fractions were observed among monoclonal antibodies, suggesting differences in epitope specificities. Three antibodies cross-reacted with B. nummifer crude venom. Two monoclonal antibodies were utilized to develop a two-site enzyme-immunoassay for myotoxin detection at the nanogram level. Three of the antibodies (one IgM and two IgG1) showed ability to neutralize myotoxicity of purified myotoxin in preincubation type experiments.

The structural gene for the ribosomal protein S18 in Escherichia coli. I. Genetic studies on a mutant having an alteration in the protein S18

Abstract A mutant of Escherichia coli strain CR341, originally isolated as a temperature-sensitive mutant, was found to have an altered 30 S ribosomal protein (S18) in addition to and independently of temperature sensitivity. Protein S18 from the mutant strain differs in electrophoretic mobility in polyacrylamide gel electrophoresis at pH 4.5 from protein S18 of the parental origin. The mutation responsible for the alteration in S18 is different from two other mutations in the mutant strain which give the temperature-sensitive phenotype. The gene involved in the S18 alteration is located in a region between 76 and 88 minutes on the E. coli genetic map; the location is outside the str-spc region at 64 minutes, where several known ribosomal protein genes are located. An episome covering the loci rha (76 min) through pyr B (84 min) was introduced into the mutant. The resultant merodiploid strains were shown to produce both the normal and the mutant forms of S18. The results support the conclusion described in the accompanying paper (Kahan et al ., 1973) that the mutation studied is in the structural gene for S18.

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

Abstract 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.

Reverse-phase high-performance liquid chromatography of Escherichia coli ribosomal small subunit proteins☆

Abstract Reverse-phase high-performance liquid chromatography has been explored as an approach for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes. The majority of these proteins are of similar molecular weight and isoelectric point, making separation by size exclusion or ion exchange difficult. With the use of an octadecasilyl silica column and a trifluoroacetic acid-acetonitrile solvent system, the 21 proteins of the 30 S subunit have been separated into 15 peaks. The yield of total protein recovered from the column was ≥85%. The proteins present in each peak have been identified by polyacrylamide gel electrophoretic analysis of the peaks as well as by comparison with the relative retention volumes of known purified 30 S proteins on the column. The results clearly show that this method is a powerful and rapid technique for the identification and purification of 30 S proteins. Analysis of [ 3 H]puromycin-labeled 30 S subunit protein provides an illustrative example of its utility for affinity labeling studies.

Protein-protein proximity in the association of ribosomal subunits of Escherichia coli: Crosslinking of 30 S protein S16 to 50 S proteins by glutaraldehyde or formaldehyde☆

The interaction of ribosomal subunits from Escherichia coli has been studied using crosslinking reagents. Radioactive 35S-labeled 50 S subunits and non-radioactive 30 S subunits were allowed to reassociate to form 70 S ribosomes. The 70 S particles, containing radioactivity only in the 50 S protein moiety, were incubated with glutaraldehyde or formaldehyde. As a result of this treatment a substantial fraction of the 70 S particles did not dissociate at 1 mm-Mg2+. This fraction was isolated and the ribosomal proteins were extracted. The protein mixture was analyzed by the Ouchterlony double diffusion technique by using eighteen antisera prepared against single 30 S ribosomal proteins (all except those against S3, S15 and S17). As a result of the crosslinking procedure it was found that only anti-S16 co-precipitated 35S-labeled 50 S protein. It is concluded that the 30 S protein S16 is at or near the site of interaction between subunits and can become crosslinked to one or more 50 S ribosomal proteins.