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M Hortsch

Publications and source records attributed to M Hortsch.

At least 19 recordsLinked to original sources
PubMed1992

Annulin, a protein expressed at limb segment boundaries in the grasshopper embryo, is homologous to protein cross-linking transglutaminases.

Annulin, a protein whose general stage- and position-specific expression pattern in the grasshopper embryo is described in the companion paper, is expressed in epithelial annuli in developing limbs. Here, we show that these annuli comprise narrow circumferential bands of epithelial cells at the boundaries of limb segments. At most boundaries, expression of annulin precedes the first morphological signs of segmentation. The most distal cells in a band underlie the boundary invagination. Bands arise in a stereotyped order and, within a band, expression occurs in an ordered circumferential progression. Annulin has a molecular weight of about 97 kDa and appears to be intracellular and peripherally associated with the inner leaflet of the cell membrane. Using the monoclonal antibody 7H7, two overlapping cDNA clones encoding this protein were isolated from an embryonic Schistocerca cDNA expression library. The nucleotide and deduced amino acid sequences indicate that the annulin protein does not contain either a signal sequence or a transmembrane domain. By sequence comparison, annulin appears to be a transglutaminase, one of a family of enzymes that have protein cross-linking activity. Its expression pattern within the limb and the embryo is associated with areas undergoing morphogenetic rearrangements, movements, or rapid cell division. It may stabilize cells under mechanical stress or participate in some other way in these morphogenetic activities.

Amino Acid Sequence↗
PubMed1991

Sticky molecules in not-so-sticky cells.

The assignment of specific roles to cell-surface proteins by standard methods can be a major problem. In the technique described below, Schneider-2 (S2) cells, an established Drosophila cell line, have been used in cell transfection and aggregation experiments. As such, they have proved to be a useful tool for the functional characterization of putative cell-adhesion molecules.

Animals↗
PubMed1990

Drosophila fasciclin I, a neural cell adhesion molecule, has a phosphatidylinositol lipid membrane anchor that is developmentally regulated.

Fasciclin I is a homophilic neural cell adhesion molecule which is regionally expressed on a subset of fasciculating axons in both the grasshopper and Drosophila embryo, suggesting a role in axonal recognition. It is also dynamically expressed on a variety of other embryonic tissues. Biochemical analysis of the fasciclin I glycoprotein from Drosophila embryonic membranes and Schneider 1 cells indicates that it is tightly associated with the lipid bilayer by a phosphatidylinositol lipid moiety. In Drosophila embryos a large fraction of fasciclin I protein has lost its membrane anchor. The ratio of this soluble form to the phosphatidylinositol-linked form changes during embryogenesis. We speculate that removal of the phosphatidylinositol lipid from the fasciclin I protein could be a mechanism to regulate its adhesive function.

Amino Acid Sequence↗
PubMed1990

Differential splicing generates a nervous system-specific form of Drosophila neuroglian.

We recently described the characterization and cloning of Drosophila neuroglian, a member of the immunoglobulin superfamily. Neuroglian contains six immunoglobulin-like domains and five fibronectin type III domains and shows strong sequence homology to the mouse neural cell adhesion molecule L1. Here we show that the neuroglian gene generates at least two different protein products by tissue-specific alternative splicing. The two protein forms differ in their cytoplasmic domains. The long form is restricted to the surface of neurons in the CNS and neurons and some support cells in the PNS; in contrast, the short form is expressed on a wide range of other cells and tissues. Thus, whereas the mouse L1 gene appears to encode only one protein that functions largely as a neural cell adhesion molecule, its Drosophila homolog, the neuroglian gene, encodes at least two protein forms that may play two different roles, one as a neural cell adhesion molecule and the other as a more general cell adhesion molecule involved in other tissues and imaginal disc morphogenesis.

Amino Acid Sequence↗
PubMed1990

Membrane biogenesis during B cell differentiation: most endoplasmic reticulum proteins are expressed coordinately.

The induction of high-rate protein secretion entails increased biogenesis of secretory apparatus organelles. We examined the biogenesis of the secretory apparatus in the B cell line CH12 because it can be induced in vitro to secrete immunoglobulin (Ig). Upon stimulation with lipopolysaccharide (LPS), CH12 cells increased secretion of IgM 12-fold. This induced secretion was accompanied by preferential expansion of the ER and the Golgi complex. Three parameters of the rough ER changed: its area and volume increased 3.3- and 3.7-fold, respectively, and the density of membrane-bound ribosomes increased 3.5-fold. Similarly, the area of the Golgi stack increased 3.3-fold, and its volume increased 4.1-fold. These changes provide sufficient biosynthetic capacity to account for the increased secretory activity of CH12. Despite the large increase in IgM synthesis, and because of the expansion of the ER, the concentration of IgM within the ER changed less than twofold during the differentiation process. During the amplification of the rough ER, the expression of resident proteins changed according to one of two patterns. The majority (75%) of rough microsomal (RM) proteins increased in proportion to the increase in rough ER size. Included in this group were both lumenal proteins such as Ig binding protein (BiP), and membrane proteins such as ribophorins I and II. In addition, the expression of a minority (approximately 9%) of RM polypeptides increased preferentially, such that their abundance within the RM of secreting CH12 cells was increased. Thus, the expansion of ER during CH12 differentiation involves preferential increases in the abundance of a few resident proteins, superimposed upon proportional increases in most ER proteins.

Animals↗
PubMed1990

Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting.

Fasciclin I is a membrane-associated glycoprotein that is regionally expressed on a subset of fasciculating axons during neuronal development in insects; it is expressed on apposing cell surfaces, suggesting a role in specific cell adhesion. In this paper we show that Drosophila fasciclin I is a novel homophilic cell adhesion molecule. When the nonadhesive Drosophila S2 cells are transfected with the fasciclin I cDNA, they form aggregates that are blocked by antisera against fasciclin I. When cells expressing fasciclin I are mixed with cells expressing fasciclin III, another Drosophila homophilic adhesion molecule, the mixture sorts into aggregates homogeneous for either fasciclin I- or fasciclin III-expressing cells. The ability of these two novel adhesion molecules to mediate cell sorting in vitro suggests that they might play a similar role during neuronal development.

Animals↗
PubMed1990

Drosophila neurotactin, a surface glycoprotein with homology to serine esterases, is dynamically expressed during embryogenesis.

Drosophila neurotactin is a transmembrane glycoprotein with an apparent molecular mass of 135 x 10(3). Neurotactin is regionally expressed at the cellular blastoderm stage; later in embryogenesis the expression of the protein becomes restricted to cells of the peripheral and central nervous system. Immunocytochemical localization shows neurotactin protein at points of cell-cell contact. Using the anti-neurotactin monoclonal antibody BP-106, a neurotactin cDNA was isolated that encodes a 846 residue polypeptide. The chromosomal location of the neurotactin gene is 73C. The extracellular domain at the carboxyterminal end of the neurotactin protein shows a strong structural and sequence homology to serine esterases without retaining the amino acids forming the active center. Neurotactin therefore belongs to a growing group of proteins including Drosophila glutactin and thyroglobulins that are known to share this serine esterase protein domain motif without retaining the active center of the enzyme.

Amino Acid Sequence↗
PubMed1989

Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1.

Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.

Amino Acid Sequence↗
PubMed1988

The human docking protein does not associate with the membrane of the rough endoplasmic reticulum via a signal or insertion sequence-mediated mechanism.

Docking protein (DP, or SRP receptor) is an essential component of the cellular machinery that mediates the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (ER). In this study we have investigated the nature of its own targeting to its site of function, the rough ER. Using an in vitro transcription-translation system we demonstrate that DP is not inserted into the membrane via a classical SRP/DP-mediated process (in contrast to human ribophorins), nor via hydrophobic insertion sequences (in contrast to cytochrome b5). Instead, we suggest that membrane assembly of DP is receptor-mediated; requiring the presence in the membrane of other proteins that mediate its targeting and insertion.

Animals↗
PubMed1988

A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum.

We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.

Animals↗
PubMed1987

Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins.

Ribophorins I and II represent proteins that are postulated to be involved in ribosome binding. They are abundant, highly-conserved glycoproteins located exclusively in the membranes of the rough endoplasmic reticulum. As the first step in the further characterization of the structure and function of these proteins, we have isolated and sequenced full-length human cDNA clones encoding ribophorins I and II using probes derived from a human liver expression library cloned into pEX1. The authenticity of the clones was verified by overlaps in the protein sequence of N-terminal and several internal fragments of canine pancreatic ribophorins I and II. The cDNA clones hybridize to mRNA species of 2.5 kb in length, and encode polypeptides of 68.5 and 69.3 kd, respectively. Primary sequence analysis, coupled with biochemical studies on the topology, indicates that both ribophorins are largely luminally disposed, spanning the membrane once and having 150 and 70 amino acid long cytoplasmically disposed C termini, respectively. Both are synthesized as precursors having cleavable signal sequences of 23 (ribophorin I) and 22 (ribophorin II) amino acids. The topology suggested by the primary structure has been confirmed biochemically using proteolytic enzymes and anti-ribophorin antibodies. Proteolysis of intact microsomes with a variety of enzymes resulted in a reduction in the apparent mol. wt of ribophorin I that would correspond to a loss of its 150-amino acid cytoplasmic tail. In the case of ribophorin II, it is completely resistant to such proteolysis which is consistent with its luminal disposition and fairly hydrophobic C terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Binding Sites↗
PubMed1985

A structural and functional analysis of the docking protein. Characterization of active domains by proteolysis and specific antibodies.

Docking protein is a 73-kDa integral membrane protein of the rough endoplasmic reticulum. It is essential for translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Monoclonal and polyclonal antibodies have been generated which, in conjunction with limited proteolysis, have been used to characterize several subspecies of docking protein. These proteolytic fragments have been analyzed with respect to the various functions ascribed to docking protein which can be assayed in vitro. Proteolytic digestion of membrane-associated or of affinity-purified intact docking protein showed that: elastase cleavage generates a 59-kDa soluble fragment and one of 14 kDa which contains the membrane anchoring domain; trypsin as well as endogenous proteolysis generates a 46-kDa fragment, leaving a 27-kDa domain containing the membrane anchor. This 27-kDa fragment can be reduced to a 13- and a 14-kDa piece by elastase digestion. The characteristics of these various subspecies were examined. The 59-kDa soluble fragment, which can reconstitute full translocation activity to docking protein-depleted microsomes (Meyer, D. I., and Dobberstein, B. (1980) J. Cell Biol. 87, 503-508) was capable of releasing a signal recognition particle-mediated translation arrest. The 46-kDa fragment was neither able to reassociate with nor to reconstitute the activity of docking protein-depleted microsomes. Moreover this fragment was unable to release a signal recognition particle-mediated arrest. This suggests that the 13-kDa fragment (the difference between 46 and 59 kDa) is both essential for association with the membrane, and for the release of translation arrests.

Animals↗