Assembly Competence Research Articles

Expression of a human alpha-tubulin: properties of the isolated subunit.

We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts. Both systems produce soluble, full-length human alpha-tubulin polypeptide. When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures. The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation. Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer. Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer. In contrast, alpha-tubulin expressed in E. coli lysates was incapable of coassemblying with bovine brain tubulin. Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit. These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies [Sherman, G., Rosenberry, T.L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156] which imply that this residue is in a salt-bridge interaction in the dimer.

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than ;mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.

Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form.

Pro-OmpA that is synthesized in vitro can assemble into bacterial inner membrane vesicles in the presence of ATP and NADH. We have purified pro-OmpA to determine which additional soluble proteins are necessary for its membrane assembly. [35S]Pro-OmpA was bound to Sepharose-linked antibody to OmpA, then eluted with 8 M urea and chromatographed on an anion-exchange resin in 8 M urea. This pro-OmpA is purified 2000-fold and is radiochemically pure. After dialysis, it is soluble but incompetent for membrane assembly. Addition of an Escherichia coli cytoplasmic fraction (S100) to the assembly reaction does not allow translocation. However, when S100 is added to pro-OmpA prior to dialysis, full assembly competence is restored, suggesting that a soluble factor, termed "trigger factor," triggers the folding of pro-OmpA into an assembly-competent form as the urea is removed. We noted that, prior to the last purification step, the immunoaffinity-purified pro-OmpA was partially competent for membrane assembly without addition of trigger factor. To test whether trigger factor had bound to the antibody column by means of its association with pro-OmpA, the crude pro-OmpA was acid-denatured prior to immunoadsorption. In this experiment, the trigger factor did not bind to the anti-OmpA column, and S100 was required for renaturation of this [35S]pro-OmpA. As suggested by this experiment, the crude [35S]pro-OmpA was in a complex with other proteins. Sedimentation velocity studies showed that the trigger factor has an apparent molecular weight of approximately 60,000. We propose that it is required for translocation-competent folding of pro-OmpA and other precursor proteins.

Microtubules in immature oocytes of Xenopus laevis.

Previous work indicated that immature oocytes of Xenopus were incapable of assembling microtubules but that competence was achieved during maturation. We report here that small numbers of microtubules do exist in immature oocytes. Consistent with this finding, ultrastructural observations indicate that brain microtubules injected into immature oocytes persist in large numbers for at least 30 min. We report that the tubulin dimers of mature and immature oocytes are equally capable of assembling with brain tubulin in vitro. We confirmed previous results that injection of taxol into immature oocytes has no effect when assayed by light microscopy. However, ultrastructural observations suggest that some microtubule assembly is stimulated by taxol. We tested for the ability of immature oocytes to elongate microtubules from 'seeds' by injecting deciliated pellicles of Tetrahymena. No elongation was observed either by light or electron microscopic observation. We conclude that the immature oocyte is capable of very limited microtubule assembly and that a marked increase in assembly competence occurs during maturation. Our data suggest that the change in assembly competence during maturation is due to the release, activation or synthesis of a stimulatory co-factor.

Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate.

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.

The Effects of X Irradiation on Microtubule Assembly in Vitro

Microtubular proteins isolated from beef brains were x-irradiated (500 to 30,000 rad) in the assembled or unassembled state in virto, and the effects of radiation on the samples were examined. Samples were irradiated in a water bath at 0 (ice slurry), 22 or 24, and 37/sup 0/C. The assembly of microtubules was monitored by following changes in optical density at 340 nm and/or changes in specific viscosity, combined with electron microscopy of the samples at various stages of experimentation. Samples containing microtubles were 30 (22/sup 0/C) to 50% (37/sup 0/C) more sensitive to inactivation by irradiation than samples containing unassembled microtubular proteins. Doses between 500 and 2000 rad had little effect on the assembly competency of the microtubular proteins. Doses of 5000 to 30,000 rad reproducibility inhibited the assembly of microtubules. More than 80% of the observed inactivation of microtubular proteins by 30,000 rad can be accounted for by the oxidation of critical sulfhydryls.

Endogenous particulate inhibitor of microtubule assembly in developing mammalian brain

Microtubule assembly in vitro was inhibited by an endogenous particulate fraction of mouse brain. This inhibitory activity was isolated from the supernatant obtained when assembled microtubules were separated from soluble tubulin by centrifugation of crude brain extracts in glycerol medium at room temperature. The inhibitory fraction could be pelleted upon dilution of glycerol. The endogenous particulate fraction inhibited microtubule assembly in a concentration-dependent manner, and in a manner similar to soluble yeast RNA and other polyanions. The factor was sensitive to RNase and heat. The amount of this factor, relative to tubulin levels, varied with neonatal animal age in a manner which appears to explain our earlier observation ( J. A. Koehn, and R. W. Olsen, 1978, Arch. Biochem. Biophys., 186, 114–120) of an age variation in tubulin assembly competence. Variations in the assembly properties of tubulin itself did not appear to explain these age differences. Whereas extracts obtained at room temperature in microtubule-stabilizing medium containing glycerol appeared to have a greater fraction of assembled tubulin in neonatal brain than in adult, soluble extracts obtained at 0 °C without glycerol showed no age-dependent differences in their overall assembly competency in vitro. The action of this RNA-containing, heat-sensitive particulate factor on microtubule assembly may reflect an in vivo regulatory mechanism.