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Biomedical subjects

M T Haeuptle

Publications and source records attributed to M T Haeuptle.

8 recordsLinked to original sources
PubMed1989

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane.

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.

Amino Acid Sequence↗
PubMed1989

Structural requirements for membrane assembly of proteins spanning the membrane several times.

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.

Cell Membrane↗
PubMed1987

Signal recognition particle arrests elongation of nascent secretory and membrane proteins at multiple sites in a transient manner.

The signal recognition particle (SRP) has been shown to target nascent secretory and membrane proteins to the endoplasmic reticulum. In the wheat germ cell-free system, SRP arrests the elongation of the nascent chains until the translational complex is docked to the endoplasmic reticulum membrane where the interaction between SRP and docking protein causes a release of the nascent chain arrest. For two secretory proteins, arrested peptides of 70 amino acids have been identified (Walter, P., Ibrahimi, I., and Blobel, G. (1981) J. Cell Biol. 91, 545-550; Meyer, D. I., Krause, E., and Dobberstein, B. (1982) Nature 297, 647-650). By using an in vitro coupled transcription-translation system, we have analyzed SRP arrest and the resulting peptides of the two secretory proteins lysozyme and granulocyte-macrophage colony-stimulating factor and the membrane protein invariant chain. SRP arrested the elongation of all three proteins at multiple sites, giving rise to ladders of arrested peptides. The size of the arrested peptides increased with the time of translation, resulting in mostly full-length pre-peptides after about 40 min. This suggests that SRP arrest in transient rather than stable. Upon addition of microsomes, the SRP arrest was released, and all the blocked peptides could be chased into mature proteins or full-length precursors.

Animals↗
PubMed1986

Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length.

We investigated the arrest of mRNA translation at predetermined sites by oligodeoxynucleotides complementary to defined coding sequences within a mRNA. An in vitro transcription and a wheat germ cell-free translation system were used for the synthesis of mRNA and protein, respectively. Oligodeoxynucleotides (10-, 15- and 20-mer) arrested polypeptide synthesis in a concentration-dependent manner at the site of their hybridization to the mRNA, as judged by the size of the translation products. A 5-mer oligodeoxynucleotide did not prevent synthesis of the full length protein. Ribosomes arrested by an oligodeoxynucleotide transiently stacked up and eventually disassembled. Upon dissociation of the ribosomes from the blocked site, nascent chains were released as peptidyl-tRNAs which in turn became rapidly converted to free polypeptide chains. None of these results was affected by the position within the reading frame to which the 3' end of the oligodeoxynucleotide hybridized. The general applicability of translation arrest by oligodeoxynucleotides was demonstrated for different mRNAs. Only partial arrest of translation was obtained when oligodeoxynucleotides were used to arrest translation in the reticulocyte cell-free system.

Animals↗
PubMed1983

Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture.

We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and alpha-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with the collagen gels. Storage and secretion of fat, caseins and alpha-lactalbumin required the presence of PRL, whereas the accumulation and vectorial discharge of transferrin was prolactin independent. There was no differentiation gradient between the tip and the cent of the outgrowth, since DNA synthesis and milk protein storage were random along the tubular structures. These results indicate that establishment of functional polarity and induction of cytodifferentiation are influenced by the nature of the interaction of the cells with the collagen structure. The morphological differentiation in turn plays an important role in the synthesis, storage, and secretion of fat and milk proteins.

Animals↗
PubMed1983

Cell proliferation and milk protein gene expression in rabbit mammary cell cultures.

We analyzed the synthesis of DNA, the rate of cell proliferation, and the expression of milk protein genes in mammary cells grown as primary cultures on or in collagen gels in chemically defined media. We assessed DNA synthesis and cell growth, measured by [(3) H]- thymidine incorporation into acid-insoluble material, DNA content, and cell counts, in a progesterone- and prolactin-containing medium. In some experiments, cultures were pulsed for 1 h with [(3)H]thymidine and dissociated into individual cells which were cytocentrifuged and processed for immunocytochemistry and autoradiography. We analyzed expression of milk protein genes at the transcriptional, translation and posttranslational levels in progesterone-depleted medium in the presence or absence of prolactin. We measured protein secretion by radioimmunoassays with antisera directed against caseins, alpha-lactalbumin and milk transferrin1. We determined protein synthesis by incorporating radio-labeled amino acids into acid-precipitable material and by immunoprecipitating biosynthetically labeled milk proteins. We assessed the accumulation of casein mRNA by hybridizing total cellular RNA extracted from cultured cells with (32)P-labeled casein cDNA probes. On attached collagen gels, the cells synthesized DNA and replicated until they became confluent. The overall protein synthetic activity was low, and no milk proteins were synthesized or secreted even in the presence of prolactin. The block in milk protein gene expression was not restricted to translational or posttranslational events but also included transcription, since no casein mRNA accumulated in these cells. On floating gels, protein synthesis was threefold higher than in cells from attached gels. Overall protein synthesis as well as casein and alpha-lactalbumin synthesis and secretion were prolactin-dependent with maximal stimulation at around 10(-9) M. A marked inhibition occurred at higher hormone concentrations. Casein mRNA accumulated in these cells, provided prolactin was present in the medium. In contrast, these cells did not synthesize DNA, nor did they replicate. In embedding gels, the rate of cell proliferation was exponential over 25 d with a doubling time of approximately 70 h. The overall protein synthesis increase was parallel in time with the increase in cell number. Caseins and alpha-lactalbumin (in contrast to transferrin) were synthesized only in the presence of prolactin. We observed the same hormone dependency as with cells growing on floating gels. The number of casein- and transferring-positive cells was measured after dissociating the cell cultures. At day 12, 60 percent of the total cells stored transferring in small cytoplasmic vesicles, whereas only 25 percent of the cells accumulated casein. Differences in the organization and in the shape of mammary cells depending on cell surface conditions suggest that the geometry of the cells, their interaction with extracellular matrix constituents, and cell-to-cell interactions play a role in the expression of two mammary functions: DNA synthesis and growth, as well as milk protein gene expression.

Animals↗