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D Louvard

Publications and source records attributed to D Louvard.

At least 145 records · Page 8Linked to original sources
PubMed1982

Characterization of molecules involved in protein translocation using a specific antibody.

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.

Animals↗
PubMed1982

Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport.

Frozen, thin sections of baby hamster kidney (BHK) cells were incubated with either concanavalin A (Con A) or Ricinus communis agglutinin I (RCA) to localize specific oligosaccharide moieties in endoplasmic reticulum (ER) and Golgi membranes. These lectins were then visualized using an anti-lectin antibody followed by protein A conjugated to colloidal gold. All Golgi cisternae and all ER membranes were uniformly labeled by Con A. In contrast, RCA gave a uniform labeling of only half to three-quarters of those cisternae on the trans side of the Golgi stack; one or two cis Golgi cisternae and all ER membranes were essentially unlabeled. This pattern of lectin labeling was not affected by infection of the cells with Semliki Forest virus (SFV). Infected cells transport only viral spike glycoproteins from their site of synthesis in the ER to the cell surface via the stacks of Golgi cisternae where many of the simple oligosaccharids on the spike proteins are converted to complex ones (Green, J., G. Griffiths, D. Louvard, P. Quinn, and G. Warren. 1981. J. Mol. Biol. 152:663-698). It is these complex oligosaccharides that were shown, by immunoblotting experiments, to be specifically recognized by RCA. Loss of spike proteins from Golgi cisternae after cycloheximide treatment (Green et al.) was accompanied by a 50% decrease in the level of RCA binding. Hence, about half of the RCA bound to Golgi membranes in thin sections was bound to spike proteins bearing complex oligosaccharides and these were restricted to the trans part of the Golgi stack. Our results strongly suggest that complex oligosaccharides are constructed in trans Golgi cisternae and that the overall movement of spike proteins is from the cis to the trans side of the Golgi stack.

Animals↗
PubMed1980

Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells.

A dog kidney epithelial cell line (MDCK), grown in monolayer, displayed in vitro an asymmetric localization of surface proteins. Aminopeptidase [alpha-aminoacylpeptide hydrolase (microsomal), EC 3.4.11.2] was found only in the apical face whereas Na+, K+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was found in the basolateral faces. These two faces are delineated by the junctional complex at which close cell-cell contact occurs. alpha-Actinin, a protein associated with plasma membranes, was concentrated near the region of cell-cell contact. When membrane proteins in the apical surface were crosslinked and subsequently removed from the surface by endocytosis, crosslinked antigens reappeared in the apical face at the region of cell-cell contact. Antigens that were not crosslinked were also (re)inserted in the same region. This process was not affected by cycloheximide, presumably because a large pool of apical membrane proteins (observed in small cytoplasmic vesicles) was used to replace the endocytosed antigens. It is psoposed that the region containing the junctional complex is involved in guiding apical membrane proteins to their final location.

Actinin↗
PubMed1980

Interactions between the plasma membrane and cytoskeleton of cultured fibroblasts.

Observations of cultured cells made by double-fluorescence staining indicate that regions of the plasma membrane which are in close contact with actin-containing cytoplasmic fibers have characteristics different from other regions of the membrane. On fixed cells it is found that several integral membrane proteins are excluded from these regions of membrane-fiber apposition. If, however, these same integral proteins are clustered by their specific antibodies, the patches produced are rapidly lined up over the cytoplasmic fibers, resulting in a transmembrane linkage of clustered membrane proteins to the actin cytoskeleton. These observations have led us to predict the existence of a class of integral membrane proteins, X proteins, which are associated with actin fibers and are responsible for both the initial exclusion and then the transmembrane linkage of clusters of other integral membrane proteins. In an attempt to identify X proteins we have produced antisera against purified plasma membranes prepared from porcine intestinal brush borders. These antisera detect surface antigens on fixed human fibroblast cells which are initially lined up over actin fibers. These antigens are, thus, candidates for the hypothesized X proteins. Using the fluorescence microscope, we are attempting to isolate the potential X antigens with a staining absorption assay.

Actins↗
PubMed1978

Transmembrane interactions and the mechanisms of transport of proteins across membranes.

We have made observations, by double fluorescence staining of the same cell, of the distributions of surface receptors, and of intracellular actin and myosin, on cultured normal fibroblasts and other flat cells, and on lymphocytes and other rounded cells. The binding of multivalent ligands (a lectin or specific antibodies) to a cell surface receptor on flat cells clusters the cell receptors into small patches, which line up directly over the actin- and myosin-containing stress fibers inside the cell. Similar ligands binding to rounded cells can cause their surface receptors to be collected into caps on the surface, and these caps are invariably found to be associated with concentrations of actin and myosin under the capped membrane. Although these ligand-induced surface phenomena appear to be different on flat and rounded cells, we propose that in both cases clusters of receptors become linked across the membrane to actin- and myosin-containing structures. In flat cells these structures are very long stress fibers; therefore, when clusters of receptors become linked to these fibers, the clusters are immobilized. In round cells, membrane-associated actin- and myosin-containing structures are apparently much less extensive than in flat cells; therefore, clusters of receptors linked to these structures are still mobile in the plane of the membrane. We suggest that in this case the clusters are then actively collected into a cap by an analogue of the muscle sliding filament mechanism. To explain the transmembrane linkage, we propose that actin is associated with the plasma membrane as a peripheral protein which is directly or indirectly bound to an integral protein (or proteins) X of the membrane. Individual molecules of any receptor are not bound to X, but after they are specifically clustered into patches, a patch of receptors then becomes bound to S and hence to actin/myosin.

Actins↗
PubMed1977

Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts.

The surface distributions of three different membrane integral proteins, beta2-microglobulin (part of the histocompatibility antigen complex), aminopeptidase (alpha-aminoacyl-peptide hydrolase; EC 3.4.11.2), and the Na+,K+-ATPase (ATP phosphohydrolase; EC 3.6.1.3) on human fibroblasts grown in monolayer culture have been studied with their specific antibodies by immunofluorescence. On the same cells, the distribution of intracellular actin was observed by a spectrally distinct fluorescent staining procedure. If each of the antibody reagents was permitted to cluster its specific protein in the plane of the membrane, these clusters apparently became linked, through the membrane, to actin- and myosin-containing filaments (stress fibers) underneath the membrane, and were thereby immobilized. From these and other experiments, it appears that most, if not all, integral proteins can, upon clustering, form such transmembrane linkages to actin and myosin. A molecular mechanism for the formation of these linkages is proposed which postulates that actin is associated with the cytoplasmic surface of plasma membranes by peripheral attachment to a ubiquitous integral protein X in the membrane; when other integral proteins are induced to form clusters, they become bound to X and hence to actin (and myosin). The possible physiological role of these transmembrane linkages is briefly discussed.

Actomyosin↗
PubMed1976

Structural and topological homology between porcine intestinal and renal brush border aminopeptidase.

A method for the preparation of closed, right-side-out vesicles from the brush border membrane of the kidney proximal tubules is described. The aminopeptidase known to be bound to this membrane was investigated in order to compare its properties with those already reported for the intestinal enzyme. Both are composed of a hydrophilic, catalytically active part lying on the external side of the membrane and a short hydrophobic domain probably located in the N-terminal region of one of the subunits ensuring fixation to the lipid matrix. The enzyme were also found to be clinically similar. Moreover, a quantitative immunological technique showed that they contained 6 cross-reacting determinants, consistent with a very high degree of homology. Four of these determinants were accessible in the bound form of the enzymes in the region of the active site. The other two, probably related to the junction between the hydrophilic moiety and the hydrophobic anchor were completely masked in the bound form. The remainder (6 in the intestinal and 4 in the renal enzyme), were heterologous. The accessibility of two well determinants in this latter group was substantially reduced, perhaps by the proximity of the lipid and/or of other enzyme molecules.

Amino Acids↗
PubMed1976

On the hydrophobic part of aminopeptidase and maltases which bind the enzyme to the intestinal brush border membrane.

The intestinal brush border aminopeptidase and unfractionated maltases M2+M3 are composed of a hydrophilic, sugar containing and enzymatically active part, and a smaller hydrophobic part presumably binding the catalytic part of the lipid matrix of the membrane. Hydrophobic parts detaced by trypsin from the detergent forms of aminopeptidase and the maltases were purified and shown to have molecular weights ranging from 8000 to 10000. All are rich in hydrophobic residues and contain no disulfide bridges. However, their overall amino acid composition is different. The hydrophobic parts appear to be N-terminal in the detergent forms of the enzymes.

Amino Acids↗