Structural changes in tubulin sheets upon removal of microtubule-associated proteins

Zinc-induced tubulin sheets without microtubule-associated proteins (MAPs) were assembled from tubulin purified by phosphocellulose chromatography. Large, open sheets were obtained in five-minute incubations at pH 5.7. Electron micrographs of negatively stained sheets showed a protofilament arrangement similar to that observed for zinc-induced sheets with MAPs but with altered lattice parameters. The spacings measured from optical diffraction patterns demonstrated that the protofilaments were 2.2 A closer together in the sheets without MAPs. Each MAP-free sheet was also divided roughly in half by a discontinuity which was parallel to the protofilaments and the relationship between the two domains was deduced from computed transforms. Two-dimensional image processing was carried out by conventional Fourier techniques and by correlation analysis. The correlation analysis improved the reconstructions in this application, with the resolution limited by the inherent properties of the negative stain method to about 14 A. A prominent feature of the computed reconstructions was an alternation of light and dark protofilaments due to differential staining, as revealed by a study of folded sheets. Neighboring protofilaments are related by a 2-fold screw axis, as they are in zinc-induced sheets with MAPs, but the symmetry is masked by the differential staining. The major effect of MAP removal on the structure of the sheets is that the bilobed structure of alternate tubulin subunits is no longer observed. This observation and the closer spacing of protofilaments is consistent with the postulate that some of the MAP molecules lie in the groove between protofilaments and bind to several tubulin dimers.     

Structural studies on porcine hemopexin

1. Porcine hemopexin was isolated from the serum of a single animal and purified to homogeneity. 2. Porcine hemopexin has an apparent Mw of 67,000, binds heme in a 1:1 molar ratio and consists of 24% N-linked oligosaccharides. The amino acid composition of porcine hemopexin compares well with the amino acid composition of human and rabbit hemopexins. 3. Limited tryptic hydrolysis of apohemopexin generates stable peptides of apparent Mw 42,000, 25,000, 24,000 and 21,000. The tryptic peptide of apparent Mw 42,000 (peptide I) binds heme in a 1:1 molar ratio, consists of 33% N-linked oligosaccharides and is derived from the amino terminal of intact hemopexin. The three peptides of smaller-Mw (collectively peptide II) represent the carboxyl terminal half of hemopexin, do not contain N-linked oligosaccharides and have no heme-binding capability. The Mw heterogeneity of peptide II is likely due to cleavage at secondary sites. 4. Under nondissociating electrophoresis two bands are resolved for hemopexin and peptide I, indicating the possibility of polymorphism in porcine hemopexin.     

33K protein--an inhibitory factor of tubulin polymerization in porcine brain

A factor (33K protein) that modulates tubulin polymerization in vitro has been purified to homogeneity from porcine brain by ammonium sulfate fractionation and Whatman DE52, Toyo-pearl HW65C and Bio-Gel A 0.5 m column chromatographies. The purified fraction was free of nucleic acids and sugars. The activity of the purified 33K protein is pronase E sensitive but apparently heat- and trypsin-resistant though it undergoes tryptic digestion. The 33K protein inhibits polymerization of brain microtubule proteins in a dose-dependent manner and partially depolymerizes preformed microtubules. It also inhibits polymerization of purified starfish tubulin and microtubule elongation involving fragellar outer doublet microtubules and purified porcine brain tubulin. This suggests that the target of the 33K protein is tubulin rather than microtubule-associated proteins. The 33K protein causes incomplete depolymerization of microtubules and a new steady state is quickly attained which is apparently independent of microtubule mass concentration. Divalent cations such as calcium and magnesium do not modulate the inhibitory activity of the 33K protein.     

Structural studies on dissociated porcine gastric mucus gel

Reduction of the insoluble porcine mucus gel with dithiothreitol, followed by molecular sieve chromatography, resulted in the recovery of the two fragments of molecular size 2.54 X 10(6) and 1.75 X 10(6). Both gastric mucus glycoproteins have been cleaved by pronase, papain or trypsin into fragments whose molecular sizes range from 5.60 X 10(5) to 1.87 X 10(6) daltons. On reduction, the enzyme-treated fragments were further dissociated into smaller fragments which have been characterized by SDS-PAGE. The evidence indicates that the pol: neric structure of gastric mucus differs from that of mucus of varying origins, in particular with respect to the size and number of component subunits per native molecule.     

Structure of the tubulin dimer in zinc-induced sheets

The structure of tubulin has been studied in projection by minimum beam electron microscopy and image processing of negatively stained zinc-induced sheets. The reconstructed images include data to 15 Å resolution.We report here a clear and reproducible 82 Å repeat arising from the arrangement of heterodimers in sheet aggregates of tubulin. This repeat is only observed in diffraction patterns from images recorded by minimum beam methods (10 to 20 e/Ų) and arises from small, but consistent, structural differences between two similar subunits believed to represent the two chemical species of tubulin monomer (M_r, 55,000). At higher electron doses (100 to 200 e/Ų), the additional information is lost or very much reduced, and only a repeat of 41 Å is observed, owing to the loss of distinction between monomers in the tubulin heterodimer.The sheets are composed of 49 Å wide, polar protofilaments, similar to those observed in microtubules; however, the interprotofilament packing is completely different in the two structures. In these sheets, adjacent protofilaments point and face in opposite directions; i.e. they are related by dyad-screw axes normal to the protofilament axes and in the plane of the sheet. Thus, the zinc-induced sheets are crystals of space group P2₁, with cell dimensions of about 97 Å × 82 Å, containing one tubulin heterodimer per asymmetric unit.Reconstructed images of four individual sheets, and their average, show the arrangement and shapes of the two heterodimers contained in each unit cell. The structure and packing of heterodimers in sheets are compared to those in opened out microtubules where all protofilaments point and face in the same direction.     

Three-dimensional reconstruction of tubulin in zinc-induced sheets.

The three-dimensional structure of porcine brain tubulin in planar sheets formed in the presence of zinc has been determined to a resolution of approximately 20 Å by electron microscopy and image reconstruction on negatively stained samples. The samples were prepared with a mica floatation technique, which yields tubulin sheets with 36 reciprocal space maxima on lattice lines at 21, 28, 42 and 84 Å^?1 in Fourier transforms of digitized images. In order to obtain three-dimensional data, sheets were tilted with the goniometer stage of the electron microscope to provide images at various angles between 0 ° and ± 60 °. Transforms of 33 tilted images plus the transform of untilted sheets based on an average of nine untilted images were combined to give the third dimension of reciprocal space ($\text{z}{}^1$). These data, were expressed in terms of the phases and amplitudes along the $\text{z}{}^1$ lattice line for each of the 36 maxima observed in untilted samples, as well as five additional lattice lines which have zero-amplitudes in the non-tilted central section of the three-dimensional transform. Home of these zero-amplitudes arise from systematic absences which are due to a 2-fold screw axis relating adjacent protofilaments of tubulin in the zinc-induced sheets. Thus in the three-dimensional reconstructions of the sheets a polarity of the protofilaments is apparent, with adjacent protofilaments aligned in opposite directions to give an antiparallel pattern, in contrast to normal microtubules composed of protofilaments in parallel alignment. Two classes of morphological units, each with a mass corresponding to a molecular weight of about 55,000, are found to alternate along the protofilaments. These distinct morphological units are identified as the α and β subunits of tubulin, confirming the representation of tubulin as an αβ heterodimer. Furthermore, the extensive internal contact between subunits within a dimer can readily be distinguished from the less extensive contact between dimer units. Such differences in contacts were not apparent in the earlier two-dimensional reconstructions. In addition, areas of excluded stain joining one class of subunits to the subunits of the other class in adjacent protofilaments have been resolved for tubulin polymerized in zinc-induced sheets. Of the two classes of subunits one is distinguished by a prominent cleft. Identification of which class of subunits is α and which is β is not yet possible.     

Phenobarbital induces aneuploidy in Saccharomyces cerevisiae and stimulates the assembly of porcine brain tubulin

Phenobarbital (PB) specifically induces mitotic chromosomal malsegregation in the diploid Saccharomyces cerevisiae strain D61.M but no other genetic events such as mitotic recombination or point mutations. In accordance with the hypothesis that PB exerts its genotoxic activity by an interaction with tubulin, it stimulates the GTP-promoted assembly of porcine brain tubulin in vitro. This process is reversible thus excluding an unspecific denaturation of the tubulin protein by PB.     

Bacteriospermia in extended porcine semen

Bacteriospermia is a frequent finding in freshly extended porcine semen and can result in detrimental effects on semen quality and longevity if left uncontrolled. The primary source of bacterial contamination is the boar. Other sources that have been identified include environment, personnel, and the water used for extender preparation. A 1-year retrospective study was performed on submissions of extended porcine semen for routine quality control bacteriological screening at the University of Pennsylvania. Out of 250 sample submissions, 78 (31.2%) tested positive for bacterial contamination. The most popular contaminants included Enterococcus spp. (20.5%), Stenotrophomonas maltophilia (15.4%), Alcaligenes xylosoxidans (10.3%), Serratia marcescens (10.3%), Acinetobacter lwoffi (7.7%), Escherichia coli (6.4%), Pseudomonas spp. (6.4%), and others (23.0%). Prudent individual hygiene, good overall sanitation, and regular monitoring can contribute greatly in controlling bacterial load. Strategies that incorporate temperature-dependent bacterial growth and hyperthermic augmentation of antimicrobial activity are valuable for effective control of susceptible bacterial loads. Aminoglycosides remain the most popular antimicrobial class used in porcine semen extenders, with beta-lactam and lincosamide use increasing. With the advent of more novel antimicrobial selection and semen extender compositions in swine, prudent application and understanding of in vitro pharmacodynamics are becoming paramount to industry success in the use of this breeding modality.     

Structural basis of tubulin tyrosination by tubulin tyrosine ligase

Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL–tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton.     

Human brain tubulin purification: Decrease in soluble tubulin with age

The soluble tubulin of human cerebral cortex, as assessed by [³H]colchicine binding of the 100,000g supernatant fraction, decreases drastically with age, 75 percent from age 0 to age 90. There is also a considerably lower concentration of high molecular weight proteins in the soluble fraction of postmortem human cerebral cortex than in that of nonhuman species. Human brain tubulin can be polymerized into microtubules with DEAE-dextran. The DEAE-dextran induced microtubules are stable to cold temperature (4°) and calcium. However, in the presence of 1 M glutamate, the microtubules become cold labile and depolymerize at 4°. Thus we have developed a novel method for purifying polymerization competent tubulin from fresh or frozen human cerebral cortex. Human brain tubulin purified by our novel method is very similar to tubulin from the brains of other mammals in molecular weight, amino acid composition, polymerization-depolymerization parameters, and structural dimensions of the microtubules formed.Some aspects of this work have been published as an abstract in 1981. Fed. Proc. 40:1548.     

Structural rearrangements in tubulin following microtubule formation

Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the alpha beta-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation--as found in zinc-induced tubulin sheets--and an outward curved conformation--as found in tubulin-stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by beta-tubulin, whereas alpha-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.     

Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.     

Structural and functional domains of tubulin

The molecular aspects of the microtubule system is a research area that has developed very rapidly during the past decade. Research on the assembly mechanisms and chemistry of tubulin and the molecular biology of microtubules have advanced our understanding of microtubule formation and its regulation. The emerging view of tubulin is of a macromolecule containing spatially discrete sequences that constitute functionally different domains with respect to self-association, interactions with microtubule associated proteins (MAPs) and specific ligands. Recent studies point to the role of the carboxyl-terminal moiety of tubulin subunits in regulating its assembly into microtubules. These investigations combined with further studies on the spatial relationships between tubulin domains should provide new insights into the detailed structural basis of microtubule assembly.     

N-terminally extended form of C-type natriuretic peptide (CNP-53) identified in porcine brain

C-type natriuretic peptide of 22 residues (CNP-22) is very recently identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system newly established for CNP-22, we searched for CNP-related peptides in porcine brain. In addition to CNP-22, one major form of immunoreactive CNP was detected in porcine brain extracts, being isolated by immunoaffinity chromatography and reverse phase high performance liquid chromatography. By microsequence analysis, the peptide was deduced to be a 53-amino acid peptide carrying a CNP-22 sequence at the C-terminus, and was designated C-type natriuretic peptide-53 (CNP-53). CNP-53 was found to be a major molecular form of CNP in porcine brain.     

Projection map of tubulin in zinc-induced sheets at 4 A resolution.

Tubulin polymerizes into two-dimensional, crystalline sheets in the presence of zinc ions. These sheets are well suited to structural studies by electron crystallography. We have developed conditions for forming sheets which are large and well ordered enough to provide both electron diffraction and image data to better than 4 A resolution. In projection maps calculated from this data, the alpha and beta monomers can be identified within the protofilaments. These results indicate that we should be able to determine the structure of tubulin in these sheets at atomic resolution.     

Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.