Delineation of functional regions within the subunits of the Saccharomyces cerevisiae cell adhesion molecule a-agglutinin.
a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on the surface of cells of a mating type and consists of an anchorage subunit Aga1p and a receptor binding subunit Aga2p. Cell wall attachment of Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C., Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We report here that purified Aga2p was unstable and had low molar specific activity relative to its receptor alpha-agglutinin. Aga2p co-expressed with a 149-residue fragment of Aga1p formed a disulfide-linked complex with specific activity 43-fold higher than Aga2p expressed alone. Circular dichroism of the complex revealed a mixed alpha/beta structure, whereas Aga2p alone had no periodic secondary structure. A 30-residue Cys-rich Aga1p fragment was partially active in stabilization of Aga2p activity. Mutation of either or both Aga2p cysteine residues eliminated stabilization of Aga2p. Thus the roles of Aga1p include both cell wall anchorage and cysteine-dependent conformational restriction of the binding subunit Aga2p. Mutagenesis of AGA2 identified only C-terminal residues of Aga2p as being essential for binding activity. Aga2p residues 45-72 are similar to sequences in soybean Nod genes, and include residues implicated in interactions with both Aga1p (including Cys(68)) and alpha-agglutinin.
Department of Biological Sciences and the Institute for Biomolecular Structure and Function, Hunter College of the City University of New York, New York 10021, USA.
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Shen
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Wang
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Pike
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Jue
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Zhao
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de Nobel
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Kurjan
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Lipke
D000373:Agglutinins / Q000737:chemistry / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002473:Cell Wall / Q000502:physiology; D003001:Cloning, Molecular; D004220:Disulfides; D000072235:Mating Factor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000373:Agglutinins; D004220:Disulfides; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D000072235:Mating Factor