The immunological and molecular characterization of alpha-agglutinin from Saccharomyces cerevisiae.
Item
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Title
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The immunological and molecular characterization of alpha-agglutinin from Saccharomyces cerevisiae.
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Identifier
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AAI9108190
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identifier
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9108190
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Creator
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Wojciechowicz, Donald Charles.
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Contributor
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Adviser: Peter N. Lipke
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Date
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1990
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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}\alpha{dollar}-agglutinin is a cell surface glycoprotein expressed on mating type {dollar}\alpha{dollar} cells in the yeast Saccharomyces cerevisiae. The function of {dollar}\alpha{dollar}-agglutinin is to bind to a-agglutinin, thereby causing agglutination of cells of opposite mating type. Agglutination between cells of opposite mating type enhances the probability of cell fusion to form a diploid zygote. Therefore, {dollar}\alpha{dollar}-agglutinin is an important cell adhesion molecule which facilitates mating in Saccharomyces cerevisiae.;The work presented here further clarifies the structure, function and expression of {dollar}\alpha{dollar}-agglutinin. An polyclonal antibody was used to study the cell surface expression of {dollar}\alpha{dollar}-agglutinin. I have shown that there are 5 {dollar}\times{dollar} 10{dollar}\sp4{dollar} molecules of {dollar}\alpha{dollar}-agglutinin constitutively expressed per {dollar}\alpha{dollar} cell. Induction with a-factor causes a modest increase in {dollar}\alpha{dollar}-agglutinin expression to 6.5 {dollar}\times{dollar} 10{dollar}\sp4{dollar} molecules per cell. The spatial distribution of {dollar}\alpha{dollar}-agglutinin on the cell surface in uninduced cells is polar while induced cells express {dollar}\alpha{dollar}-agglutinin more uniformly. Buds and daughter cells do not express cell surface {dollar}\alpha{dollar}-agglutinin. Lastly, {dollar}\alpha{dollar}-agglutinin is expressed in the shmoo tip region of induced {dollar}\alpha{dollar} cells, the location were a and {dollar}\alpha{dollar} cells fuse to form a diploid zygote.;Using the antibody, I have also shown that the {dollar}AG\alpha{dollar}1 gene codes for {dollar}\alpha{dollar}-agglutinin. This gene is {dollar}\alpha{dollar}-specific and shown to be required for agglutinability of {dollar}MAT\alpha{dollar} cells. Notable features of {dollar}AG\alpha{dollar}1 include that the ORF (1) codes for a 70 kD protein; (2) contains an acidic region within the amino half of the glycoprotein; (3) contains a 15 amino acid hydrophobic carboxy terminus; (4) has a putative N and O-linked glycosylation sites throughout the coding sequence.;Phenotypes of {dollar}AG\alpha{dollar}1 truncation mutants further elucidate the domain structure of {dollar}\alpha{dollar}-agglutinin. Using molecular cloning to introduce termination codons within the ORF of {dollar}AG\alpha{dollar}1, I have shown that (1) the carboxy hydrophobic terminus of {dollar}\alpha{dollar}-agglutinin is necessary for cell wall anchorage and (2) the amino half of the glycoprotein (possibly the acidic region) contains the binding domain of {dollar}\alpha{dollar}-agglutinin. Most of the O-linked sugar is associated with the carboxy half of the molecule while N-linked carbohydrate is found throughout {dollar}\alpha{dollar}-agglutinin. Lastly, I show that {dollar}\alpha{dollar}-agglutinin is efficiently transported through the secretory pathway.
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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.
