A pathway for cell wall anchorage of alpha-agglutinin from Saccharomyces cerevisiae.
Item
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Title
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A pathway for cell wall anchorage of alpha-agglutinin from Saccharomyces cerevisiae.
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Identifier
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AAI9510686
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identifier
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9510686
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Creator
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Lu, Chafen.
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Contributor
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Adviser: Peter N. Lipke
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Molecular | Health Sciences, Immunology
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Abstract
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{dollar}Saccharomyces\ cerevisiae\ \alpha{dollar}-agglutinin is a cell wall-anchored adhesion glycoprotein. It can be released from the cell wall by {dollar}\beta{dollar}-glucanase treatment. The predicted {dollar}\alpha{dollar}-agglutinin polypeptide contains a potential signal sequence for glycosyl phosphatidylinositol (GPI) anchor addition. It was shown in this study that the {dollar}\alpha{dollar}-agglutinin was synthesized with a GPI anchor. Two GPI-anchored intermediate forms were identified: the 140 kD ER form and the {dollar}>{dollar}300 kD plasma membrane form. GPI linkage to these forms was demonstrated by susceptibility to PI-PLC cleavage and metabolic labeling with ({dollar}\sp3{dollar}H) myo-inositol and ({dollar}\sp3{dollar}H) palmitic acid. A soluble form of {dollar}>{dollar}300 kD that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the cell wall-anchored form. Additional forms of 80 kD, 150 kD and 250-300 kD were detected in temperature-sensitive secretory mutants. The 80 kD form is likely to represent the unmodified {dollar}\alpha{dollar}-agglutinin peptide; the 150 kD and 250-300 kD forms were membrane-bound and are likely to be intermediates between the 140 kD ER form and the {dollar}>{dollar}300 kD plasma membrane form. N- and O-glycosylation, and probably other modifications resulted in successive increases in size during transport to the cell surface.;The mature cell wall {dollar}\alpha{dollar}-agglutinin, but not the intermediate forms, was immunoreactive with antibodies against {dollar}\beta{dollar}-1,6-glucan. The cell wall {dollar}\alpha{dollar}-agglutinin from kre mutants, which have reduced size of the cell wall {dollar}\beta{dollar}-1,6-glucan, exhibited lower molecular size and less immunoreactivity with the anti-{dollar}\beta{dollar}-1,6-glucan. These observations demonstrate that the cell wall {dollar}\alpha{dollar}-agglutinin is covalently associated with the cell wall {dollar}\beta{dollar}-1,6-glucan. A C-terminal 29 amino acid truncated form of {dollar}\alpha{dollar}-agglutinin, which was secreted into the medium due to a defect in GPI anchor addition, did not contain {dollar}\beta{dollar}-1,6-glucan, suggesting a role for the GPI anchor in cross-linking of {dollar}\alpha{dollar}-agglutinin and {dollar}\beta{dollar}-1,6-glucan.;The results constitute the first experimental support for a novel cell wall anchorage mechanism: the GPI anchor acts to localize {dollar}\alpha{dollar}-agglutinin to the plasma membrane and the cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate, followed by cross-linkage to the wall {dollar}\beta{dollar}-glucan.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
