Biomedical subjects
S M Hurtley
Publications and source records attributed to S M Hurtley.
Lysosomal degradation of ubiquitin-tagged receptors.
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Capturing genes encoding membrane and secreted proteins important for mouse development.
A strategy based on the gene trap was developed to prescreen mouse embryonic stem cells for insertional mutations in genes encoding secreted and membrane-spanning proteins. The "secretory trap" relies on capturing the N-terminal signal sequence of an endogenous gene to generate an active beta-galactosidase fusion protein. Insertions were found in a cadherin gene, an unc6-related laminin (netrin) gene, the sek receptor tyrosine kinase gene, and genes encoding two receptor-linked protein-tyrosine phosphatases, LAR and PTP kappa. Analysis of homozygous mice carrying insertions in LAR and PTP kappa showed that both genes were effectively disrupted, but neither was essential for normal embryonic development.
Membrane proteins involved in targetted membrane fusion.
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Hot line to the secretory pathway.
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Recycling of a secretory granule membrane protein after stimulated secretion.
Recycling of a secretory granule membrane protein, dopamine-beta-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-beta-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-beta-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-beta-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized antibody could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.
Now you see it, now you don't: the Golgi disappearing act.
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Clathrin- and non-clathrin-coated vesicle adaptors.
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Protein translocation into reconstituted vesicles.
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Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP).
We have characterized the association between the binding protein, BiP (also known as GRP 78), and misfolded forms of the influenza virus hemagglutinin precursor, HA0. BiP is a heat-shock-related protein that binds to unassembled immunoglobulin heavy chain and to a variety of misfolded proteins in the lumen of the ER. A small fraction (5-10%) of newly synthesized HA0 in CV-1 cells was found to be misfolded and retained in the ER. When glycosylation was blocked with tunicamycin, all of the HA0 produced was similarly misfolded. The misfolded HA0 was retained as relatively small (9-25-S) complexes associated with BiP. In these complexes the top domains of HA0 were correctly folded judging by their reactivity with monoclonal antibodies, but the polypeptides were cross-linked via anomalous interchain disulfides. The association with BiP was non-covalent and easily broken by warming to 37 degrees C or by adding ATP to the lysate. Pulse-chase experiments showed that HA0's self-association into complexes occurred immediately after synthesis and was followed rapidly by BiP association. The misfolded, BiP-associated HA0 was not transported to the plasma membrane but persisted as complexes in the ER for a long period of time before degradation (t1/2 = 6 h). The results suggested that BiP may be part of a quality control system in the ER and that one of its functions is to detect and retain misfolded proteins.
Protein oligomerization in the endoplasmic reticulum.
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Reconstitution of an endocytic fusion event in a cell-free system.
Using a cell-free system we have obtained fusion of vesicles from the endocytic pathway. The fusion is rapid, efficient, and requires ATP. Only vesicles derived from certain positions along the endocytic pathway are capable of fusing. Lysosomes and vesicles derived from the plasma membrane do not fuse.