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Biomedical subjects

M Rüdiger

Publications and source records attributed to M Rüdiger.

At least 37 records · Page 2Linked to original sources
PubMed1998

Suppression of tubulin tyrosine ligase during tumor growth.

The C terminus of the tubulin alpha-subunit of most eukaryotic cells undergoes a cycle of tyrosination and detyrosination using two specific enzymes, a tubulin tyrosine ligase (TTL) and a tubulin carboxypeptidase. Although this enzyme cycle is conserved in evolution and exhibits rapid turnover, the meaning of this modification has remained elusive. We have isolated several NIH-3T3 derived clonal cell lines that lack TTL (TTL-). TTL- cells contain a unique tubulin isotype (delta2-tubulin) that can be detected with specific antibodies. When injected into nude mice, both TTL- cells and TTL- cells stably transfected with TTL cDNA form sarcomas. But in tumors formed from TTL rescued cells, TTL is systematically lost during tumor growth. A strong selection process has thus acted during tumor growth to suppress TTL activity. In accord with this result, we find suppression of TTL activity in the majority of human tumors assayed with delta2-tubulin antibody. We conclude there is a widespread loss of TTL activity during tumor growth in situ, suggesting that TTL activity may play a role in tumor cell regulation.

3T3 Cells↗
PubMed1998

Yel013p (Vac8p), an armadillo repeat protein related to plakoglobin and importin alpha is associated with the yeast vacuole membrane.

Proteins of the armadillo family are involved in diverse cellular processes in higher eukaryotes. Some of them, like armadillo, beta-catenin and plakoglobins have dual functions in intercellular junctions and signalling cascades. Others, belonging to the importin-alpha-subfamily are involved in NLS recognition and nuclear transport, while some members of the armadillo family have as yet unknown functions. Here, we introduce the Saccharomyces cerevisiae protein Yel013p as a novel armadillo (arm) repeat protein. The ORF Yel013w was identified in the genome project on chromosome V (EMBL: U18530) and codes for an acidic protein of 578 residues with 8 central arm-repeats, which are closely related to the central repeat-domain of Xenopus laevis plakoglobin. We show that Yel013p (Vac8p) is constitutively expressed in diploid and haploid yeasts and that it is not essential for viability and growth. However, the vacuoles of mutant cells are multilobular or even fragmented into small vesicles and the processing of aminopeptidase I, representing the cytoplasm-to-vacuole transport pathway, is strongly impaired. Consistent with these observations, subcellular fractionation experiments, immunolocalization and expression of green fluorescent protein (GFP) fusion proteins revealed that Yel013p (Vac8p) is associated with the vacuolar membrane. Our data provide evidence for the involvement of an arm-family member in vacuolar morphology and protein targeting to the vacuole.

Amino Acid Sequence↗
PubMed1997

Characterization of two F-actin-binding and oligomerization sites in the cell-contact protein vinculin.

Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/plasma membrane junctions at cell contacts. To understand its role in organizing the distal portions of microfilaments into specific, morphologically distinct structures at these sites in more detail, we characterized its interaction with filamentous actin and with itself by means of in vitro assays. Using recombinant proteins comprising different parts of the vinculin tail fused to the maltose-binding protein of Escherichia coli, we show in sedimentation assays that this part of vinculin harbors two discrete sites that can bind to actin independently. They reside within amino acid residues 893-985 and 1016-1066 of the 1066-residue polypeptide chain. However, both sites are necessary to cross-link or bundle actin filaments, as demonstrated by low shear viscometry. Crosslinking and bundling are alternatives determined by the molar ratio of fusion protein to F-actin. Both actin-binding sequences are capable of oligomer formation, as shown in chemical-cross-linking and dot-overlay assays. These data allow us to propose a possible role for vinculin in organizing the distal ends of microfilaments at the plasma membrane into the point-like structure characteristic for cell-matrix contacts.

Actins↗
PubMed1997

Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.

The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely inactivate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues.

Anions↗
PubMed1997

Reconstitution of bacteriorhodopsin from the apoprotein and retinal studied by Fourier-transform infrared spectroscopy.

The reconstitution of the retinal-containing protein bacteriorhodopsin (BR) from the apoprotein and retinal has been studied by Fourier-transform infrared (FTIR) difference spectroscopy. 9-cis-Retinal which occupies the binding site but does not reconstitute the chromophore was used as "caged retinal". Photoisomerization to the all-trans isomer triggers the reconstitution reaction. Absorption bands in the FTIR difference spectra of the educt and product of the reaction could be assigned by comparison with a 9-cis-retinal FTIR spectrum or an FT-Raman spectrum of BR and due to band shifts observed upon deuterium exchange. Specific difference bands were assigned to the protonated carboxyl groups of D96 and D115 by use of the mutants D115N and D96N. Both aspartic acids are protonated also in the apoprotein with pKa values above 10 and undergo a frequency shift toward higher wavenumbers indicating a more hydrophobic environment in the reconstituted protein. No indication was found for protonation changes of carboxyl groups or other protonatable residues when carrying out the reaction at pH values between 4 and 10. The pH-dependent protonation changes reported earlier [Fischer & Oesterhelt (1980) Biophys. J. 31, 139-146] therefore may be caused by protons in a hydrogen-bonded network. Mutations of E204, but not of D38 or E9, cancel proton uptake during reconstitution at high pH as well as proton release at low pH. It is concluded, that E204, without changing its protonation state itself, is part of a protonatable hydrogen-bonded network which changes its pKa during reconstitution thereby causing the observed protonation changes.

Bacteriorhodopsins↗
PubMed1996

VASP interaction with vinculin: a recurring theme of interactions with proline-rich motifs.

VASP (vasodilator-stimulated phosphoprotein), a protein associated with microfilaments at cellular contact sites, has been identified as a ligand for profilin and zyxin, two proteins also involved in microfilament dynamics and organization at these regions. Here, we report that VASP also directly binds to vinculin, another component of adherens junctions. Competition experiments with a vinculin-derived peptide showed that a proline-rich motif, located in the hinge region that connects vinculin's head and tail domains, is involved in VASP binding. The same motif is present in zyxin but the interactions of VASP with vinculin and zyxin differ in detail. Hence, this motif may be recognized by VASP in different ways when presented in distinct cellular sites.

Amino Acid Sequence↗
PubMed1996

Molecular and structural analysis of a continuous birch profilin epitope defined by a monoclonal antibody.

The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 --> Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.

Allergens↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Intramolecular interactions regulate serine/threonine phosphorylation of vinculin.

Using protein kinase C, we have studied the influence of intramolecular interactions on phosphorylation in vinculin. We show that vinculin and its 90 kDa head and 29/27 kDa tail fragments, generated by V8 proteolytic cleavage, are differentially phosphorylated. While intact vinculin and the isolated head domain are only weakly labelled, the isolated tail fragment is much more strongly phosphorylated. In the presence of the tail, the head is fully protected from the kinase. These data are consistent with our observation that native vinculin is primarily phosphorylated within the tail domain and suggest a function of vinculin phosphorylation in the regulation of the vinculin conformation.

Amino Acid Sequence↗
PubMed1996

Pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase from Streptomyces arenae is closely related to GAPDH from thermostable eubacteria and plant chloroplasts.

Streptomyces arenae produces the antibiotic pentalenolactone, a highly specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). During the phase of pentalenolactone production, S. arenae expresses a pentalenolactone-insensitive GAPDH isoform; otherwise, a pentalenolactone-sensitive form is expressed. The gene of the pentalenolactone-insensitive GAPDH was cloned and sequenced. Regulatory elements typical for genes encoding antibiotic resistance and production are localized upstream and downstream of the open reading frame. No expression of pentalenolactone-insensitive GAPDH was detected in Streptomyces lividans transformed with the gene. In Escherichia coli, the gene was expressed from an induced lac promoter. Amino-terminal sequencing of the heterologously expressed GAPDH proved its identity with pentalenolactone-insensitive GAPDH from S. arenae. Sequence comparisons with GAPDH from other organisms showed a close relationship to GAPDH of plant chloroplasts, of other gram-positive bacteria, and of thermophilic gram-negative bacteria. Pentalenolactone-insensitive GAPDH differs from all closely related GAPDHs only in a few residues, none of which are directly involved in catalysis or substrate binding. The total amino acid composition is more similar to GAPDH of thermophilic species than to that of mesophilic species. The purified enzyme was moderately thermotolerant, which could be a side effect of the structural changes causing pentalenolactone-resistance.

Amino Acid Sequence↗
PubMed1996

Crosstalk between cell adhesion molecules: vinculin as a paradigm for regulation by conformation.

With the identification of ever more protein components associated with cellular adhesion sites, the nature of the mechanisms underlying assembly and maintenance of these important cellular structures was in danger of becoming completely intangible. However, new information on how the interaction between the different proteins can be regulated is beginning to shed more light on this problem. In particular, recent biochemical and electron microscopic data on the overall structure and function of vinculin, one of the key structural proteins involved in cellular adhesion, leads to a novel model for the regulation of cellular adhesion.

Journal Article↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Chemical reconstitution of a chloride pump inactivated by a single point mutation.

The arginine residue R108 plays an essential role in the transport mechanism of the light-driven anion pump halorhodopsin (HR) as demonstrated by complete inactivation of chloride transport in mutant HR-R108Q. In the presence of substrate anions, guanidinium ions bind to the mutant protein with affinities in the mM range, thereby restoring transport activity and photochemical properties of wild type. One guanidinium ion and one anion are bound per molecule of HR-R108Q. For HR wild type, HR-R108Q-guanidinium and HR-R108K, differences in transport activity and anion selectivity are found which may be explained by effects of anion solvation. The agreement between light-induced FTIR difference spectra of HR wild type and HR-R108Q-guanidinium demonstrates that no structural changes occur in the reconstituted mutant and that the photoreactions of wild type and reconstituted mutant are identical. Furthermore, an IR absorbance band of the guanidino group of R108 can be identified at 1695/1688 cm-1. In HR-R108Q, a guanidinium ion binding close to the mutated residue is proposed to mimick the role of the R108 side chain as the anion uptake site. Thus the wild type reaction mechanism is reconstituted.

Anions↗
PubMed1995

Characterization of post-translational modifications of brain tubulin by matrix-assisted laser desorption/ionization mass spectrometry: direct one-step analysis of a limited subtilisin digest.

Matrix-assisted ultraviolet laser desorption/ionization (MALDI) mass spectrometry was used to investigate the molecular masses and heterogeneity patterns caused by post-translational modifications in tubulin from porcine brain. Direct analysis of the limited digest with subtilisn shows that the molecular masses of the majority of the carboxyterminal fragments are below 2 kDa, while the truncated tubulin subunits have lost approximately the same mass. The results confirm the cleavage sites previously postulated for this protease. The mass information on the peptides allows the degree of polyglutamylation to be measured directly and shows that molecules with two glutamyl residues in the side chain are the most abundant species. In addition it identifies the degree of tyrosination of alpha tubulin. This one-step monitoring of a complex digest provides information equivalent to that obtainable from the purified components, while the amount of material required is reduced by three orders of magnitude when compared to previous studies. MALDI spectra partially resolve the alpha and beta subunits of the highly homogeneous tubulin from turkey erythrocytes, which lacks polyglutamylation but does not separate alpha and beta subunits from the heterogeneous brain tubulin. Post-translational modifications of the brain tubulin result in shifting peaks to higher molecular masses, in broadening of the peaks, and in loss of resolution.

Amino Acid Sequence↗
PubMed1995

The molecular architecture of focal adhesions.

This article outlines the present knowledge of the architecture, molecular composition, and dynamics of focal contacts of adhesive animal cells. These structures, developed at the plasma membrane at sites where cells touch their substratum, are essential for cellular attachment in tissue formation during embryogenesis and wound healing. In tissue culture, they are particularly prominent and thus amenable to detailed investigation. Focal contacts consist of a cytoplasmic face, comprising cytoskeletal elements, a transmembrane connecting region, and a extracellular face composed of proteins of the extracellular matrix. The molecular anatomy of the numerous proteins involved, the basis for classifying them as structural or regulatory components, and their in vitro interactions are described. Based on this information, current models on the dynamics of their assembly and of possible regulatory mechanisms involving a variety of signal transduction pathways are discussed.

Actins↗
PubMed1994

Intramolecular interactions in vinculin control alpha-actinin binding to the vinculin head.

Using blot overlay techniques we have investigated the interaction of vinculin with alpha-actinin. We show that an alpha-actinin binding site is located in the 90 kDa vinculin head and confirm a vinculin binding site in the C-terminal rod of alpha-actinin, as recently reported by McGregor et al. [(1994) Biochem. J. 310, 225-233]. The isolated vinculin head binds much more strongly to alpha-actinin than intact vinculin. Using a proteolytic 81 kDa head fragment, we show that vinculin residues 1-107 are required for alpha-actinin binding. Antibodies directed against vinculin residues 808-850 inhibit the vinculin-alpha-actinin binding, suggesting that this sequence is directly involved in, or topographically related to, the alpha-actinin binding site.

Actinin↗
PubMed1994

The carboxy-terminal peptide of detyrosinated alpha tubulin provides a minimal system to study the substrate specificity of tubulin-tyrosine ligase.

The ATP-dependent tubulin-tyrosine ligase (TTL) restores the carboxy-terminal tyrosine of alpha tubulin in alpha beta tubulin that has been previously detyrosinated. Here we show that the carboxy-terminal tetradecapeptide of detyrosinated alpha tubulin is used by TTL as a substrate, albeit at 50-fold lower efficiency than alpha beta tubulin. The minimal system provided by the TTL/peptide combination mirrors the TTL/tubulin system in all aspects tested, and shows a pronounced substrate inhibition. Synthetic peptides varying in length and/or containing single amino acid replacements were used to analyze the TTL specificity for the carboxy-terminal sequence of detyrosinated alpha tubulin. Peptides ending like alpha tubulin with the sequence Gly-Glu-Glu are optimally tyrosinated once a peptide length of 12 residues is reached. Position -1 of this recognition sequence, to which the tyrosine is added, must be glutamic acid. Position -2 accepts only an acidic amino acid but glutamic acid is by far preferred over aspartic acid. These results explain why a subpopulation of brain alpha tubulin, which ends with the sequence Gly-Glu, is not tyrosinated by TTL. The carboxy-terminal dodecapeptide of brain alpha tubulin with its polyglutamyl side-chain on position -6 shows the same substrate activity as the corresponding synthetic peptide lacking the side-chain. We discuss the substrate specificity of TTL for different alpha tubulins and speculate why tubulin is a better substrate than the optimal peptide covering the carboxy-terminal of detyrosinated alpha tubulin.

Animals↗