[Reconstruction of marker positions with redundant markers in gait analysis with the 3D Optotrak System].
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Equipment Design↗
Biomedical subjects
E Hartmann
Publications and source records attributed to E Hartmann.
Structurally related Spc1p and Spc2p of yeast signal peptidase complex are functionally distinct.
Two subunits of the mammalian signal peptidase complex, SPC12 and SPC25, share similar membrane topologies with the majority of each protein oriented toward the cytoplasm. Such similarities may suggest that these proteins perform redundant functions in signal peptidase activity. In the present study, we addressed this issue through analysis of the yeast homologs to SPC12 and SPC25, Spc1p and Spc2p. We show that both Spc1p and Spc2p are nonessential for signal peptidase activity and growth of yeast cells and that null mutations in the genes encoding Spc1p and Spc2p are synthetically lethal with a conditional mutation affecting Sec11p, an essential subunit of yeast signal peptidase. However, a high copy plasmid encoding Spc1p suppresses the conditional sec11 mutation, whereas the corresponding plasmid encoding Spc2p does not suppress sec11. Moreover, Spc2p, but not Spc1p, is important for signal peptidase activity and cell viability at high temperatures. These results indicate that although both Spc1p and Spc2p are noncatalytic, they are functionally distinct. Evidence is also presented that a double mutant lacking Spc1p and Spc2p grows well relative to wild type yeast cells, indicating that the signal peptidase complex missing at least two of its subunits is sufficient for signal peptidase activity in vivo.
Amino Acid Sequence↗
Importin provides a link between nuclear protein import and U snRNA export.
Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.
Amino Acid Sequence↗
The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases.
The primary structure of the 200 kDa protein of purified HeLa U5 snRNPs (U5-200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5-200kD is distinct from U5-220kD we demonstrate by protein sequencing that the human U5-specific 220 kDa protein is homologous to the yeast U5-specific protein Prp8p. A 246 kDa protein (Snu246p) homologous to U5-200kD was identified in Saccharomyces cerevisiae. Both proteins contain two conserved domains characteristic of the DEXH-box protein family of putative RNA helicases and RNA-stimulated ATPases. Antibodies raised against fusion proteins produced from fragments of the cloned mammalian cDNA interact specifically with the HeLa U5-200kD protein on Western blots and co-immunoprecipitate U5 snRNA and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co-immunoprecipitated from yeast splicing extracts containing an HA-tagged derivative of Snu246p with HA-tag specific antibodies. U5-200kD and Snu246p are thus the first putative RNA helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in vivo, indicating that the protein is essential for splicing. Anti-U5-200kD antibodies specifically block the second step of mammalian splicing in vitro, demonstrating for the first time that a DEXH-box protein is involved in mammalian splicing. We propose that U5-200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA-RNA interactions in the spliceosome.
Amino Acid Sequence↗
The homologue of mammalian SPC12 is important for efficient signal peptidase activity in Saccharomyces cerevisiae.
The multisubunit signal peptidase catalyzes the cleavage of signal peptides and the degradation of some membrane proteins within the endoplasmic reticulum (ER). The only subunit of this enzyme functionally examined to date, yeast Sec11p, is related to signal peptidase I from bacteria. Since bacterial signal peptidase is capable of processing both prokaryotic and eukaryotic signal sequences as a monomer, it is unclear why the analogous enzyme in the ER contains proteins unrelated to signal peptidase I. To address this issue, the gene encoding Spc1p, the yeast homologue to mammalian SPC12, is isolated from the yeast Saccharomyces cerevisiae. Spc1p co-purifies and genetically interacts with Sec11p, but unlike Sec11p, Spc1p is not required for cell growth or the proteolytic processing of tested proteins in yeast. This indicates that only a subset of the ER signal peptidase subunits is required for signal peptidase and protein degradation activities in vivo. Through both genetic and biochemical criteria, Spc1p appears, however, to be important for efficient signal peptidase activity.
Amino Acid Sequence↗
A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus.
The complex of importin-alpha and -beta is essential for nuclear protein import. It binds the import substrate in the cytosol, and the resulting trimeric complex moves through the nuclear pores, probably as a single entity. Importin-alpha provides the nuclear localization signal binding site, importin-beta the site of initial docking to the pore. Here we show that the conserved, basic N-terminus of importin-alpha is sufficient for importin-beta binding and essential for protein import. The fusion product of this 41 amino acid domain to a heterologous protein if transported into the nucleus in the same way as full-length importin-alpha itself. Transport is dependent on importin-beta but competed by importin-alpha. As no additional part of importin-alpha is needed for translocation, the movement which drives the import substrate complex into the nucleus appears to be generated between importin-beta and structures of the nuclear pore. The domain that binds to importin-beta appears to confer import only, but not re-export out of the nucleus, suggesting that the return of importin-alpha into the cytoplasm is not a simple reversal of its entry.
Amino Acid Sequence↗
A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae.
Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co-translational pathway of protein transport.
Amino Acid Sequence↗
Membrane topology of the 12- and the 25-kDa subunits of the mammalian signal peptidase complex.
The cleavage of signal sequences of secretory and membrane proteins by the signal peptidase complex occurs in the lumen of the endoplasmic reticulum. Mammalian signal peptidase consists of five subunits. Four have been cloned, SPC18, SPC21, SPC22/23, and SPC25, of which all but SPC25 have been demonstrated to be single-spanning membrane proteins exposed to the lumen of the endoplasmic reticulum. We have determined the cDNA sequence of the remaining 12-kDa subunit (SPC12) as well as the membrane topologies of SPC12 and SPC25 in rough microsomes. Both polypeptides span the membrane twice with their N and C termini facing the cytosol and contain only very small, if any, lumenal domains. Therefore, SPC12 and SPC25 are likely to be involved in processes other than the enzymatic cleavage of the signal sequence.
Amino Acid Sequence↗
Structure of the human CD97 gene: exon shuffling has generated a new type of seven-span transmembrane molecule related to the secretin receptor superfamily.
Recent cDNA cloning of EMR1 and CD97 suggests the existence of a new group of seven-span transmembrane (7-TM) molecules, likely encoded by a gene cluster on the short arm of chromosome 19. The membrane-spanning region of both molecules is homologous to the secretin receptor (SecR) superfamily, a group of receptors with specificity for mammalian and insect peptide hormones. Unlike members of the SecR superfamily known thus far, EMR1 and CD97 have extended extracellular regions that possess several EGF domains at the N-terminus. We herein describe the organization of the human CD97 gene, which consists of 18 exons expanding approximately 12 kb of DNA. Identical exon-intron positions in the transmembrane region indicate that the CD97 gene has evolved from an ancestral gene of the SecR superfamily. Remarkably, exons encoding the 300 amino acids by which CD97 extends the extracellular part from other members of the SecR group are preferentially separated by introns in phase 1. All three EGF domains are encoded by symmetrical class 1-1 exons, suggesting that exon shuffling to the upstream region of a precursor gene from the SecR superfamily has generated this new type of 7-TM receptor.
Antigens, CD↗
Tetracycline-regulated reporter gene expression in the moss Physcomitrella patens.
As ancestors of higher plants, mosses offer advantages as simple model organisms in studying complex processes such as development and signal transduction. Overexpression of transgenes after genetic transformation is a powerful technique in such studies. To establish a controllable expression system for this experimental approach we expressed a chimeric protein consisting of the Tn1O-encoded Tet repressor and the activation domain of Herpes simplex virion protein 16 in the moss Physcomitrella patens. We showed that this protein activates transcription from a suitable target promoter (Top 1O) containing seven operators upstream of a TATA box. In media containing very low levels of tetracycline (1 mg/l), expression levels of a beta-glucuronidase (GUS) reporter gene dropped to <1% of that in the absence of tetracycline. This regulation is due to interference of tetracycline with the DNA binding activity of the Tet repressor portion of the chimeric transcriptional activator. Stable transformants grown for three weeks on tetracycline-containing media showed negligible GUS activity, whereas GUS was expressed strongly within 24 h of transfer to tetracycline-free media. Potent and stringently regulated expression of other, physiologically active genes is thus readily available in the moss system using the convenient ToplO expression system.
Anti-Bacterial Agents↗
Differential effects of synthetic sphingosine derivatives on melanoma cell motility, growth, adhesion and invasion in vitro.
Cancer cell surface glycosphingolipids are considered to play a critical role in tumor growth and metastasis. However, the implications of glycoconjugates in the control of cell motility, which is considered to be involved in tumor invasion, are not fully understood. In this study, the effects of a series of synthetic sphingosine derivatives, obtained by the chemical transformation of azidosphingosines, on directional migration of K1735-M2 melanoma cells grown on type I collagen-coated surfaces were investigated. Following the application of 60 microM (2R, 3S, 4E)-2, 3-epimino-4-octadecen-3-ol (S4) the migration rate was 94 +/- 10 microns/day, compared with 377 +/- 22 microns/day in the control experiment. Six other analogues were not as potent. S4 also considerably down-modulated melanoma single cell motility. Inhibition of motile activity was associated with changes in the actin filament organization as well as with changes in the number and distribution of vinculin plaques. Moreover, the compound reduced the attachment abilities of melanoma cells to basement membrane Matrigel. Tumor cell invasion, however, was less affected and proliferation remained unimpaired after treatment with S4. These data suggest at least one intracellular mode of action of this particular synthetic sphingosine derivative by modulation of cytoskeletal organization. Melanoma cell motility and growth may be controlled independently via glycosphingolipids.
Actins↗
Are there similarities between the polarization of the C. elegans embryo and of an epithelial cell?
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Letter↗
Oral cadmium exposure of adults in Germany. 1: Cadmium content of foodstuffs and beverages.
The cadmium contents of 94 and 105 foodstuffs bought in six-fold repetition in 1988 and in nine-fold repetition in 1991, respectively were analysed within the framework of a market-basket study. These foodstuffs were typical of German eating habits. Additionally, 170 samples of drinking water were investigated. The cadmium concentrations of the foodstuffs were comparable with results of recent studies carried out in Europe and North America. Fruit, milk and dairy products, sugar and sugar-rich foodstuffs as well as beverages showed mean cadmium contents < or = 5 ng/g fresh matter or ng/ml, respectively. The cadmium content of meat, sausage, fish and tinned fish was also low. Pork and beef, the most important kinds of meat, contained 5.4 and 2.5 ng/g on average. The majority of the vegetables investigated, including potatoes, had cadmium concentrations < 25 ng/g. However, individuals samples of lettuce showed very high cadmium levels. The cadmium content of bread, cakes and pastries as well as farinaceous products were within the range of 20-40 ng/g. The most important bread, cakes and pastries (wheat and rye bread, toasted bread, rolls) contained 25-35 ng/g. A median cadmium concentration of 0.2 micrograms/l was found in the drinking water. As expected, liver and kidneys showed the highest cadmium levels of 73 and 204 ng/g, respectively on average.
Adult↗
Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane.
Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.
Amino Acid Sequence↗
The alignment of the axis of asymmetry in regenerating protoplasts of the moss, Ceratodon purpureus, is determined independently of axis polarity.
Ceratodon protoplasts regenerate by polar outgrowth to form cell filaments. The kinetics of regeneration show that some cellular event has to be completed before regeneration can be initiated. The development of the regeneration axis is strongly influenced by light, with axis alignment and axis polarity being fixed independently. We define axis alignment as the relationship of the regeneration axis to the incident light, independent of polarity. Thus protoplasts regenerating directly towards, or directly away from the light source are defined as being similarly aligned but with opposite polarity. Protoplasts that regenerate in unidirectional red light form axes that are aligned parallel to the light direction, with about 70% being polarised towards the light and about 30% away. In unidirectional blue or white light, almost all protoplasts regenerate towards the light but axis alignment is determined less stringently. Re-orientation of protoplasts regenerating in unidirectional light shows that axis alignment is fixed between 8 and 9 hours before protoplasts regenerate and that axis polarity is fixed later. When protoplasts are removed from directional light to either non-directional light or to darkness, regeneration axes continue to be aligned by the earlier directional stimulus for at least 24 hours. Thus although axis alignment is fixed only about 8 hours before regeneration, in the absence of contradictory information about directionality in the light environment, protoplasts retain a memory of light direction for much longer. However, both reorientation and removal from a directional light field have profound effects on axis polarity; the pattern observed in undisturbed protoplasts being lost. To account for these observations, we propose that separate gradients are established independently to determine the alignment and polarity of the regeneration axis respectively. The alignment gradient is established rapidly and is steeper in red than in blue or white light, the polarity gradient is established slowly and is steeper in white or blue light than in red. These studies will now allow a genetic dissection of these processes in moss.
Bryopsida↗
Distinct functions for the two importin subunits in nuclear protein import.
The import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs), energy and soluble factors, namely importin-alpha (M(r) 60K), importin-beta (90K) and Ran. Importin-alpha is primarily responsible for NLS recognition and is a member of a protein family that includes the essential yeast nuclear pore protein SRP1p (ref. 16). As the first event, the complex of importin-alpha and importin-beta binds the import substrate in the cytosol. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-beta. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-alpha in the nucleus. In contrast, importin-beta accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-beta on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.
Adenosine Triphosphate↗
Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretion receptor superfamily with an unusual extracellular domain.
CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and a single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).
Amino Acid Sequence↗
Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p.
We have reproduced the posttranslational mode of protein translocation across the endoplasmic reticulum membrane with reconstituted proteoliposomes containing a purified complex of seven yeast proteins. This Sec complex includes a heterotrimeric Sec61p complex, homologous to that in mammals, as well as all other membrane proteins found in genetic screens for translocation components. Efficient posttranslational translocation also requires the addition of lumenal Kar2p (BiP) and ATP. The trimeric Sec61p complex also exists as a separate entity that, in contrast with the large Sec complex, is associated with membrane-bound ribosomes. We therefore hypothesize that distinct membrane protein complexes function in co- and posttranslational translocation pathways.
Adenosine Triphosphate↗