Incorporation of nitrotyrosine into alpha-tubulin by recombinant mammalian tubulin-tyrosine ligase.

Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated alpha-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein. Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained. Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro. Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K(i) of 0.24 mM. The TTL-catalysed incorporation of nitrotyrosine as sole substrate into alpha-tubulin was clearly detectable at concentrations of 10 microM by immunological methods using nitrotyrosine specific antibodies. However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident. Analysis of the C-terminal peptides of in vitro modified alpha-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL. In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified alpha-tubulin.

Synthetic peptides identify the minimal substrate requirements of tubulin polyglutamylase in side chain elongation.

The minimal sequence requirement of Crithidia tubulin polyglutamylase is already fulfilled by tubulin-related peptides carrying a free alpha-carboxylate on a glutamic acid residue. Since the product of each glutamylation step fulfills the substrate requirements necessary for the next cycle, very long side chains are generated with brain tubulin as a substrate. Up to 70 mol of glutamic acid was incorporated per alphabeta-heterodimer. We speculate that the strict choice of a particular glutamate residue for the formation of the isopeptide bond initiating a novel side chain is made by a tubulin monoglutamylase which requires the entire tubulin as substrate.

Alpha-tubulins of Tritrichomonas mobilensis are encoded by multiple genes and are not posttranslationally tyrosinated.

Reverse-transcriptase polymerase chain reaction cloning of the 3'-ends of the alpha-tubulin cDNAs of Tritrichomonas mobilensis in combination with Southern-blot analysis identified seven to eight distinct alpha-tubulin genes. All seven lack a carboxy-terminal tyrosine and the corresponding sequence compatible with posttranslational tyrosination. This indicates that whereas tyrosination of alpha-tubulin has been found in most species, including humans and trypanosomes, it is absent in trichomonads.

Posttranslational modifications of trichomonad tubulins; identification of multiple glutamylation sites.

The alpha- and beta-tubulins present in cytoskeletons of Tritrichomonas mobilensis are extensively glutamylated. Automated sequencing and mass spectrometry of the carboxyterminal peptides identifies 4 glutamylation sites in alpha- and 2 sites in beta-tubulin. They are marked by asterisks in the terminal sequences GDE*E*E*E*DDG (alpha) and EGE*E*DEEAEA (beta). This is the first report that tubulin glutamylation can occur at multiple sites. Although T. mobilensis has four flagellae the tubulins lack polyglycylation. Thus glycylation is not necessary for formation or function of axonemal microtubules. Alpha-tubulin is completely acetylated at lysine 40 and shows no tyrosine cycle. Peptide sequences establish two distinct beta-tubulins.

Characterisation of two articulins, the major epiplasmic proteins comprising the membrane skeleton of the ciliate Pseudomicrothorax.

Most protists possess a unique membrane skeleton, the epiplasm, which is involved in pattern forming processes of the cell cortex and functions in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm. We have isolated cDNAs encoding the two major articulins of the ciliate Pseudomicrothorax dubius. Peptide sequence data confirm the identity of the cloned cDNAs encoding articulins 1 and 4. With the data presented here sequence information for all major articulins of ciliates as well as the distantly related euglenoids is available. Sequence comparison of the two newly characterised ciliate articulins with the previously determined sequences of p60, a minor articulin of the same species, and the two euglenoid articulins reveals general sequence principles and uncovers new features of this protein family. The hallmark of articulins is a central core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. These VPV-motif repeats are either 12-residues, or in some places, six residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. The sequences of these short repeats are very similar in articulins of the same species but are not conserved between euglenoids and ciliates.

Mammalian sperm tubulin: an exceptionally large number of variants based on several posttranslational modifications.

Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The alpha- and beta-tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in alpha and 0 to 15 in beta. Corresponding values for side-chain glutamyl residues were 0-6 in alpha and 0-3 in beta. Additional alpha variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in alpha- and beta-tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.

Subpellicular and flagellar microtubules of Trypanosoma brucei are extensively glutamylated.

To determine the spectrum of tubulin variants in cytoskeletons of Trypanosoma brucei carboxy-terminal fragments of alpha- and beta-tubulin were isolated and characterized by sequencing and mass spectrometry. All variants arise by posttranslational modifications. We confirm the presence of tyrosinated and detyrosinated alpha-tubulin. Unexpectedly, but in line with its sequence, beta-tubulin also occurs with and without its carboxy-terminal tyrosine. Both tyrosinated and detyrosinated alpha- and beta-tubulins are extensively glutamylated. Unglutamylated tubulins are only trace components of the cytoskeletal microtubules. The maximal numbers of glutamyl residues in the lateral chain are 15 and 6 for alpha- and beta-tubulin, respectively. The oligoglutamyl side chain is linked via an isopeptide bond to glutamic acid residues 445 of alpha- and 435 of beta-tubulin. The same sites are used in glutamylated tubulins of mammalian brain. No tubulin variants based on polyglycylation are detected in cytoskeletal preparations or in isolated flagella. Tubulin specific incorporation of radioactive glutamate but not of glycine is observed when protein biosynthesis is completely inhibited in Trypanosoma cells. Possible reasons for the absence of polyglycylated tubulins from the trypanosomal axoneme are discussed. Finally we show that lysine 40 of the flagellar alpha-tubulin is completely acetylated.

Purification and characterization of basal apparatuses from a flagellate green alga.

Basal apparatuses consisting of two basal bodies and several attached fibers were isolated from the naked green flagellate Spermatozopsis similis by detergent extraction and mechanical disintegration. Sucrose density centrifugation yielded highly enriched basal apparatuses as shown by electron microscopy. SDS-PAGE revealed the absence of histones, indicating the removal of nuclear contaminations from the isolated basal apparatuses. A mass spectrometric analysis of the carboxyterminal peptides of alpha tubulin documented detyrosination and glutamylation as posttranslational modifications and showed that some 5% of the alpha tubulin carries a polyglutamyl side chain which can reach at least 17 residues in length. Monoclonal antibodies raised against the purified basal apparatuses were used to characterize novel components in the basal apparatus. A 210-kD component identified by mAB BAS (basal apparatus of Spermatozopsis) 1.4 was localized in the flagellar transitional region by immunogold electron microscopy. Antibody BAS 16.4 reacted with two high molecular weight bands (approximately 265 and 240 kD) in Western blotting and decorated a fiber attached to the proximal end of the basal bodies. Immunofluorescence staining of isolated cytoskeletons with these mABs demonstrated that the antigens are also present in the basal apparatuses of Chlamydomonas reinhardtii and Dunahella bioculata. These antibodies are useful tools for the molecular cloning of components from the basal apparatus.

Posttranslational modifications of alpha- and beta-tubulin in Giardia lamblia, an ancient eukaryote.

Tubulin of Giardia lamblia, a representative of the oldest eukaryotes, was screened for posttranslational modifications. Mass spectrometry of the carboxy-terminal peptides documents a large number of variants. Both alpha- and beta-tubulin show polyglycylation with up to 20 and 15 extra glycyl residues respectively. Minor variants show a low level of glutamylation without or with glycylation. The glutamylation-specific antibody GT335 detects alpha- and beta-tubulin in immunoblots. The terminal tyrosine is fully retained in alpha-tubulin, which is completely acetylated at Lys-40. Thus except for the detyrosination/tyrosination cycle all posttranslational modifications known for higher eukaryotes are already present in Giardia.

Polyglycylation of tubulin in the diplomonad Giardia lamblia, one of the oldest eukaryotes.

We have searched for post-translational modifications in tubulin of the diplomonad Giardia lamblia, which is a representative of the earliest branches in eukaryotic evolution. The carboxyterminal peptide of alpha-tubulin was isolated and characterized by automated sequencing and mass spectrometry. Some 60% of the peptide is unmodified, while the remainder shows various degrees of polyglycylation. The number of glycyl residues in the lateral side chain ranges from 2 to 23. All peptide species encountered end with alanine-tyrosine, indicating the absence of a detyrosination/tyrosination cycle. We conclude that tubulin-specific polyglycylation could be as old as tubulin and axonemal structures.

The A and B tubules of the outer doublets of sea urchin sperm axonemes are composed of different tubulin variants.

The alpha beta-tubulin heterodimer, the structural unit of microtubules, comes in many variants. There are different alpha and beta isotypes encoded by multigene families. Additional heterogeneity is generated by a set of posttranslational modifications. Detyrosination of alpha-tubulin, removal of the carboxy-terminal Glu-Tyr dipeptide of alpha-tubulin, phosphorylation of some tubulins, polyglutamylation, and polyglycylation of alpha- and beta-tubulins all involve the acidic carboxy-terminal region. We have investigated the distribution of tubulin variants in the axonemal microtubules of sea urchin sperm flagella by immunological procedures and by direct sequence and mass spectrometric analysis of the carboxy-terminal peptides. The A and B tubules that comprise the nine outer doublets differ strongly in tubulin variants. A tubules contain over 95% unmodified, tyrosinated alpha beta-tubulin. In B tubules, alpha-tubulin is approximately 65% detyrosinated and both alpha- and beta-tubulin are 40-45% polyglycylated. These results show a segregation of tubulin variants between two different axonemal structures and raise the possibility that posttranslational modifications of tubulins reflect or specify structurally and functionally distinct microtubules.

Characterisation of neurofilament protein NF70 mRNA from the gastropod Helix aspersa reveals that neuronal and non-neuronal intermediate filament proteins of cerebral ganglia arise from separate lamin-related genes.

The neuronal cytoplasmic intermediate filament (IF) protein HeNF70 of the gastropod Helix aspersa is identified by sequence analysis of the corresponding 4,600 bp cDNA isolated from a cerebral ganglion cDNA library. HeNF70 shares 60% sequence identity with the neuronal LoNF70 protein of the cephalopod Loligo pealei and only 36% identity with the Helix non-neuronal IF-A protein. All three molluscan IF proteins display the lamin-type extended coil 1b subdomain harbouring six additional heptads and all have long C-terminal sequences with substantial homology to the tail domains of nuclear lamins. The lamin-like tail domains of the two neurofilament proteins share a unique motif comprising 13 residues, which is absent from Helix IF-A and all other known non-neuronal IF proteins. HeNF70 is encoded by a 9.5 kb RNA transcript. The very long 7.2 kb 3'-untranslated sequence contains a unique 26 nucleotide repeat extending over 0.5 kb in its 5'-region. The HeNF70 mRNA is expressed at low abundancy in cerebral ganglia but not in any of the 13 non-neuronal tissues tested. In contrast, all tissues express at fairly high levels the same 4.6 and 4.2 kb mRNAs encoding the Helix non-neuronal IF-A/B proteins. Blot hybridisation studies on genomic DNA and ganglion mRNA with subprobes from the cloned HeNF70 cDNA, as well as sequence analysis of an RT-PCR generated partial cDNA encoding a putative HeNF60 protein, indicate at least two different neuronal IF genes in Helix.

Purification and biochemical characterization of myomesin, a myosin-binding and titin-binding protein, from bovine skeletal muscle.

We report a method for isolating homogeneous myomesin from mammalian skeletal muscle. The identity of the purified bovine protein was confirmed by its reactivity with myomesin-specific monoclonal antibodies and with polyclonal antibodies raised against peptides derived from the amino-terminal and carboxy-terminal ends of the sequence predicted by the human myomesin cDNA. All partial sequences obtained from bovine myomesin can be aligned along the human sequence predicted by its cloned cDNA. Electron microscopy of myomesin revealed short flexible rods with a molecular length of about 50 nm. Circular dichroism spectra showed a high degree of beta structure as expected for a member of the immunoglobulin superfamily of proteins. Alignment of the sequences of the class I and II domains of myomesin with the sequences of domains of known three-dimensional structure provides a more detailed model of myomesin. In agreement with this view, the cleavage sites observed by limited proteolysis locate primarily between individual domains. In a solid-phase overlay assay myomesin specifically bound to the myosin rod and to light meromyosin (LMM), but not to the carboxy-terminal 30-kDa fragment of LMM. The myosin-binding site seemed to be confined to the amino-terminal 240 residues of the molecule. The cross-reactivity of myomesin with the phosphorylation-dependent monoclonal neurofilament antibody NE14 [Shaw, G.E., Debus, E. & Weber, K. (1984) Eur. J. Cell Biol. 34, 130-136] was analyzed. NE14 reactivity of myomesin was abolished by alkaline phosphatase. Reactivity of the antibody on stable proteolytic fragments of myomesin showed that the phosphorylation site must reside within the carboxy-terminal 60 residues.

Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization.

The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.

Beta tubulin of bull sperm is polyglycylated.

Carboxy-terminal fragments of alpha and beta tubulin from bull sperm were isolated and characterized by automated sequencing and mass spectrometry. About 60% of sperm alpha tubulin is polyglycylated. The lateral chain, which can reach 13 residues in length, is covalently attached via an isopeptide bond. The fully detyrosinated sperm alpha tubulin lacks polyglycylation. Thus mammalian sperm microtubules differ from the ciliary axonemal microtubules of the protozoan Paramecium for which others have documented a complete polyglycylation of both alpha and beta tubulin.

Primary structure of the neuronal clathrin-associated protein auxilin and its expression in bacteria.

The protein auxilin is a coat component of brain clathrin-coated vesicles. It interacts directly with the heavy chain of clathrin and supports its assembly into regular cages [Ahle, S. & Ungewickell, E. (1990) J. Cell Biol. 111, 19-29]. The combined open reading frames of three cow brain cDNA clones with a total of 4531 nucleotides predict a molecular mass of 99,504 Da for auxilin. The coding region is followed by a very long untranslated region of at least 1670 nucleotides. By Northern analysis, auxilin transcripts are found only in brain tissue. Auxilin is not related to any of the previously sequenced clathrin-binding proteins, but the region of positions 50-350 is 29% identical (similarity 56%) to the corresponding region of the actin-binding protein tensin from chicken fibroblasts. Recombinant auxilin expressed in and purified from bacteria by affinity chromatography is functional with respect to clathrin binding.

Band 6 protein, a major constituent of desmosomes from stratified epithelia, is a novel member of the armadillo multigene family.

Desmosomes are intercellular adhering junctions characteristic of epithelial cells. Several constitutive proteins--desmoplakin, plakoglobin and the transmembrane glycoproteins desmoglein and desmocollin--have been identified as fundamental constituents of desmosomes in all tissues. A number of additional and cell type-specific constituents also contribute to desmosomal plaque formation. Among these proteins is the band 6 polypeptide (B6P). This positively charged, non-glycosylated protein is a major constituent of the plaque in stratified and complex glandular epithelia. Using an overlay assay we show that purified keratins bind in vitro to B6P. Thus B6P may play a role in ordering intermediate filament networks of adjacent epithelial cells. To characterize the structure of B6P in the desmosome we have isolated cDNA clones representing the entire coding sequence. The predicted amino acid sequence of human B6P shows strong sequence homology with a murine p120 protein, which is a substrate of protein tyrosine kinase receptors and of p60v-src. P120 and B6P show amino-terminal domains differing distinctly in length and sequence. These are followed in both proteins by 460 residues that display a series of imperfect repeats corresponding to the repeats in the cadherin binding proteins armadillo, plakoglobin and beta-catenin. Over this repeat region B6P and p120 share 33% sequence identity (54% similarity). These sequence characteristics define B6P as a novel member of the armadillo multigene family and raise the question of whether the structural proteins B6P, plakoglobin, beta-catenin and armadillo share some function. Since armadillo, plakoglobin, beta-catenin and p120 seem involved in signal transduction this may also hold for B6P. The amino-terminal region of B6P (residues 1 to 263) shows no significant homology to any known protein sequence. It may therefore be involved in unique functions of B6P.

Purification of Golgi adaptor protein 1 from bovine adrenal gland and characterization of its beta 1 (beta') subunit by microsequencing.

A method for the purification of the Golgi adaptor protein 1 from bovine adrenal gland tissue was devised to investigate the relationship of its beta 1 (formerly referred to as beta') subunit to known beta-type sequences. Adrenal gland tissue was chosen for this study because it yielded 2-3 times more adaptor protein 1 than a comparable preparation from bovine brain. Like its neuronal isoform, the beta 1 subunit from adrenal gland adaptor protein 1 is readily cleaved by trypsin into a 63-kDa N-terminal fragment and a 40-kDa C-terminal fragment, while the gamma subunit is largely refractory to digestion. Based on microsequencing of 167 residues from the 63-kDa fragment, we noted 11 differences to the corresponding region of the beta 2 (formerly beta) subunit of the plasma membrane adaptor protein 2, but only one difference to the corresponding region of a beta-type protein encoded by the rat cDNA clone AP105a which is supposed to be a variant of the beta 2 subunit of the plasma membrane adaptor protein 2 [Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisberg, A., Chow, E. P., Burne, C., Keen, J. H. & Davis, A. E. (1989) Proc. Natl Acad. Sci. USA 84, 8805-8809]. Alignment of 187 residues from the 40-kDa beta 1 C-terminal fragment revealed differences in 77 positions to the corresponding region of the beta 2 subunit and differences in 23 positions compared to the supposed beta 2-like protein. These findings suggest that the protein encoded by the rat cDNA clone AP105a is more closely related to the beta 1 subunit of the bovine adrenal Golgi adaptor protein 1 than to the beta 2 subunit of the rat plasma membrane adaptor protein 2.

Transduction of Ca2+ signals upon fertilization of eggs; identification of an S-100 protein as a major Ca(2+)-binding protein.

A transient increase in the level of free cytosolic Ca2+ is observed upon fertilization of the eggs of many species and is thought to represent a key event in the initiation of development. To identify components in the egg which could be involved in mediating such Ca2+ signals we searched for Ca(2+)-binding proteins in eggs of the fresh-water fish Misgurnus fossilis (loach). We show that loach eggs contain two major Ca(2+)-binding proteins which can be purified through their Ca(2+)-dependent interaction with a hydrophobic matrix. Protein sequencing revealed that the larger 18 kDa protein is calmodulin, while the smaller polypeptide of 10 kDa is a member of the S-100 protein family. This is the first report of the presence of an S-100 protein in vertebrate eggs and shows that this protein is found in two fold higher concentration than calmodulin. Since the 10 kDa protein shares 68% sequence identity with S-100 alpha from bovine brain, it can be considered as the loach homologue of mammalian S-100 alpha. During early embryonic development, de novo protein synthesis of calmodulin is observed at the earliest stages analyzed (mid-blastula), while de novo protein synthesis of the S-100 alpha homologue begins with the mid-gastrula stage.(ABSTRACT TRUNCATED AT 250 WORDS)

SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil.

The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.