Biomedical subjects

E Hartmann

Publications and source records attributed to E Hartmann.

At least 37 records · Page 2Linked to original sources

PubMed2000

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Amino Acid Sequence↗

PubMed2000

Mammalian Sec61 is associated with Sec62 and Sec63.

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.

Amino Acid Sequence↗

PubMed2000

Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA.

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the protein-conducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved.

Animals↗

PubMed2000

[Familial myoclonus-dystonia syndrome associated with panic attacks].

We report a case of autosomal dominantly inherited dystonia and panic attacks to discuss successful treatment of a common serotonergic pathology with medication. The objective analysis of the movement disorder was done by Optotrak. First we demonstrate a reduction of the myoclonus by L-5-hydroxytryptophan, which inhibits after 11 months. After changing the medication to Nefadozone, the myoclonus and the frequency of panic attacks were reduced.

Adult↗

PubMed2000

Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus.

In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one.

Animals↗

PubMed2000

Cell type-specific mRNA quantitation in non-neoplastic tissues after laser-assisted cell picking.

OBJECTIVE: Cell type-specific mRNA quantitation can be reliably performed after harvesting less than 20 cell profiles from haemalaun-stained cryosections by laser-assisted cell picking. Up to now it has been unclear to what extent these techniques can be used to analyze differential gene expression in complex tissues. METHODS: Using a rat model of experimental endotoxin priming of the lung various pulmonary cell types were microdissected from isolated perfused and ventilated rat lungs after aerosol lipopolysaccharide/interferon-gamma stimulation. RESULTS: Porphobilinogen deaminase housekeeping gene (PBGD) and nitric oxide synthase II (NOSII) mRNA in arterial endothelial cells (AEC), bronchiolar epithelial cells (BEC), alveolar septum containing monocytes/macrophages (AS+), alveolar septum without monocytes/macrophages (AS-) and intraluminar alveolar macrophages (AM) could be quantified by real-time RT-PCR. The strongest upregulation of NOSII mRNA occurred in AM, while minimal NOSII expression was detected in BEC, AS+ and AS-. In AEC NOSII mRNA was not detectable. CONCLUSION: The combination of laser microdissection and real-time RT-PCR is a valuable tool for the quantitative in situ characterization of differential gene expression within complex tissues.

Animals↗

PubMed2000

Mesenchymal proliferation with decidual-like morphology in seminal vesicles of aging mice.

Lesions characterized by spindle and epithelioid cells and nuclear progesterone receptors are described in seminal vesicles of four aging mice. The lesions of two mice also contain granular metrial gland (GMG)-like cells. The same cellular details are seen in the uterine decidual reaction and the similar urinary bladder lesion in mice, also called mesenchymal tumor. Therefore, it is hypothesized that these lesions in male accessory sex glands and the urinary bladders of aging male and female mice are of mesenchymal origin with the potential for differentiation along several pathways, leading especially to lesions with decidual-like morphology, but also to lesions which contain only spindle cells. The decidual hypothesis is further supported by the occurrence of round eosinophilic granules and focal necrosis, interpreted as a sign of regression in all these lesion types. The bilateral lesions of a fifth mouse consist of spindle cells and scar-like tissue, the latter suggesting regression, and lack epithelioid and GMG-like cells. In this case, verification of the diagnosis depends on the demonstration of progesterone receptors, absent in normal glands. Uterine decidual reactions during pregnancy are brought about by priming with progesterone/estrogen, initiation through the blastocyst, and maintenance through progesterone. Experiments by others show that priming may also occur through growth factors/growth hormone, initiation through prostaglandins, and maintenance through testosterone in mice. It is hypothesized that upon such stimulation, certain cells in male accessory sex glands and the urinary bladder, possibly derived from the Muellerian ducts or other subperitoneal tissue, appear to have the potential in mice of developing into spindle and epithelioid cells, including decidual-like cells. All published uterine decidual reactions and lesions with decidual-like morphology in other organs of mice stayed within the peritoneal coverage of their respective organ and did not metastasize despite their "anaplastic", tumor-like appearance. Thus, they should be considered non-neoplastic. It is proposed to name above lesions in male accessory sex glands and urinary bladders "mesenchymal proliferation, decidual type" or "mesenchymal proliferation, spindle-cell type", depending on their cellular characteristics.

Aging↗

PubMed1999

Stress-associated endoplasmic reticulum protein 1 (SERP1)/Ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation.

Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61alpha and Sec61beta, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

Amino Acid Sequence↗

PubMed1999

Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4.

Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.

Amino Acid Sequence↗

PubMed1999

Chloroplast SecY is complexed to SecE and involved in the translocation of the 33-kDa but not the 23-kDa subunit of the oxygen-evolving complex.

SecY is a component of the protein-conducting channel for protein transport across the cytoplasmic membrane of prokaryotes. It is intimately associated with a second integral membrane protein, SecE, and together with SecA forms the minimal core of the preprotein translocase. A chloroplast homologue of SecY (cpSecY) has previously been identified and determined to be localized to the thylakoid membrane. In the present work, we demonstrate that a SecE homologue is localized to the thylakoid membrane, where it forms a complex with cpSecY. Digitonin solubilization of thylakoid membranes releases the SecY/E complex in a 180-kDa form, indicating that other components are present and/or the complex is a higher order oligomer of the cpSecY/E dimer. To test whether cpSecY forms the protein-conducting channel of the thylakoid membrane, translocation assays were conducted with the SecA-dependent substrate OE33 and the SecA-independent substrate OE23, in the presence and absence of antibodies raised against cpSecY. The antibodies inhibited translocation of OE33 but not OE23, indicating that cpSecY comprises the protein-conducting channel used in the SecA-dependent pathway, whereas a distinct protein conducting channel is used to translocate OE23.

Amino Acid Sequence↗

PubMed1999

CRM1-mediated recycling of snurportin 1 to the cytoplasm.

Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin alpha. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.

Animals↗

PubMed1999

Initial characterization of the nascent polypeptide-associated complex in yeast.

The three subunits of the nascent polypeptide-associated complex (alpha, beta1, beta3) in Saccharomyces cerevisiae are encoded by three genes (EGD2, EGD1, BTT1). We found the complex bound to ribosomes via the beta-subunits in a salt-sensitive manner, in close proximity to nascent polypeptides. Estimation of the molecular weight of the complex of wild-type cells and cells lacking one or two subunits revealed that the composition of the complex is variable and that as yet unknown proteins might be included. Regardless of the variability, a certain balance of the subunits has to be maintained: the deletion of one subunit causes downregulation of the remaining subunits at physiological growth temperature. Cells lacking both beta-subunits are unable to grow at 37 degrees C, most likely due to a toxic effect of the alpha-subunit. Based on in vitro experiments, it has been proposed that the function of mammalian nascent-polypeptide associated complexes (NAC) is to prevent inappropriate targeting of non-secretory nascent polypeptides. In vivo, however, the lack of NAC does not cause secretion of signal-less invertase in yeast. This result and the lack of a drastic phenotype of cells missing one, two or three subunits at optimal conditions (28 degrees C, YPD-medium) suggest either the existence of a substitute for NAC or that cells tolerate or 'repair' the damage caused by the absence of NAC.

Blotting, Western↗

PubMed1999

Functional loss of E-cadherin and cadherin-11 alleles on chromosome 16q22 in colonic cancer.

Proteins of the cadherin family regulate cellular adhesion and motility and are believed to act as tumour suppressors. Previous studies have identified frequent mutation and allelic inactivation of the E-cadherin (cadherin-1) locus in diffuse gastric cancer. At least two other cadherin genes, P-cadherin (cadherin-3) and OB-cadherin (cadherin-11), have been mapped close to the E-cadherin gene on chromosome 16q22. As this region of the genome is frequently deleted in malignancy, multiple cadherin loci may be affected by losses of chromosome 16q22. The expression of mRNA transcripts from polymorphic alleles of the E-cadherin and cadherin-11 genes was examined in 30 cases of colonic, gastric, and renal carcinoma. In gastric cancer, loss of expression of one allele was restricted to the E-cadherin locus, whilst in renal carcinoma neither locus was affected. In colonic cancers, loss of expression of one E-cadherin allele was detected in 5 of 22 cases, whilst loss of a cadherin-11 allele was seen in 5 of 23 cases. This functional loss of cadherin gene expression may be due to gene deletion, inactivation or recombination. As no evidence of cadherin gene mutation was observed in the remaining transcripts, we can conclude that these two genes are only indirectly involved in the pathogenesis of colorectal cancer.

Blotting, Northern↗

PubMed1999

Phytochrome-controlled phototropism of protonemata of the moss ceratodon purpureus: physiology of the wild type and class 2 ptr-mutants

Phototropism and polarotropism in protonemata of the moss Ceratodon purpureus are controlled by the photoreceptor phytochrome. One class of phototropism mutants is characterised by growing randomly when kept for a prolonged time (5 d or longer) in unilateral red light. It was found that a subclass of these mutants grows faster than the wild type, the rate of cell division and the length of the cells being increased. This difference is found for light-grown and dark-grown filaments. It is therefore suggested that the mutant phenotype neither results from a defect in phytochrome photoconversion nor from a defect in phytochrome-gradient formation. Instead, it is possible that a factor which is involved in both signal transduction of phototropism and regulation of cell size and cell division is deregulated. If dark-grown mutant filaments are phototropically stimulated for 24 h, they show a weak phototropic response. Phototropism and polarotropism fluence-rate effect curves for mutants were flattened and shifted to higher fluence rates compared with those for the wild type. With wild-type filaments, a previously unreported response was observed. At a low fluence rate, half of the filaments grew positively phototropically, while the other half grew negatively phototropically. It seems that under these conditions, a phytochrome gradient with two maxima for the far-red-absorbing form of phytochrome (Pfr) within the cross-section of the cell is displayed by the response of the filaments. At higher fluence rates, all filaments of the wild type grew towards the light. These data and results from microbeam irradiation experiments and from phototropism studies with filaments growing within agar, indicate that light refraction plays an important role in the formation of the Pfr gradient in phototropism of Ceratodon.

Journal Article↗

PubMed1999

Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import.

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.

Acetyltransferases↗

PubMed1998

Interaction of Kar2p and Sls1p is required for efficient co-translational translocation of secreted proteins in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.

Amino Acid Sequence↗

PubMed1998

The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation.

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the beta subunit (Sec61beta) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61beta is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61beta kinetically facilitates cotranslational translocation, but is not essential for it. Using chemical cross-linking we show that Sec61beta not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61beta and SPC25 and between Sec61beta and Sec61alpha depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

Binding Sites↗

PubMed1998

Protein translocation into the endoplasmic reticulum (ER)--two similar routes with different modes.

Protein translocation into the endoplasmic reticulum (ER) occurs cotranslationally with the ribosome tightly bound at the membrane, or post-translationally. Transport of polypeptides is performed by an elaborate structure in the ER membrane consisting of numerous proteins. Both pathways have been reconstituted in vitro using proteoliposomes that contain purified components. Primary structures of these key components have been identified and recent electron microscopic data provide a first impression of the tertiary structure of the translocation apparatus. However, the precise function of most subunits of the translocation apparatus is still a mystery and little is known about structural changes in the translocation site during a transport cycle.

Biological Transport↗