Interaction between the C termini of Alg13 and Alg14 mediates formation of the active UDP-N-acetylglucosamine transferase complex.
The second step of eukaryotic N-linked glycosylation in endoplasmic reticulum is catalyzed by an UDP-N-acetylglucosamine transferase that is comprised of two subunits, Alg13 and Alg14. The interaction between Alg13 and 14 is crucial for UDP-GlcNAc transferase activity, so formation of the Alg13/14 complex is likely to play a key role ... in the regulation of N-glycosylation. Using a combination of bioinformatics and molecular biological methods, we have undertaken a functional analysis of yeast Alg13 and Alg14 proteins to elucidate the mechanism of their interaction. Our mutational studies demonstrated that a short C-terminal alpha-helix of Alg13 is required for interaction with Alg14 and for enzyme activity. Electrostatic surface views of the modeled Alg13/14 complex suggest the presence of a hydrophobic cleft in Alg14 that provides a pocket for the Alg13 C-terminal alpha-helix. Co-immunoprecipitation assays confirmed the C-terminal three amino acids of Alg14 are required for maintaining the integrity of Alg13/Alg14 complex, and this depends on their hydrophobicity. Modeling studies place these three Alg14 residues at the entrance of the hydrophobic-binding pocket, suggesting their role in the stabilization of the interaction between the C termini of Alg13 and Alg14. Together, these results demonstrate that formation of this hetero-oligomeric complex is mediated by a short C-terminal alpha-helix of Alg13 in cooperation with the last three amino acids of Alg14. In addition, deletion of the N-terminal beta-strand of Alg13 caused the destruction of protein, indicating the structural importance of this region in protein stability.
Mesh Terms:
Amino Acid Sequence, Animals, Glycosylation, Humans, Magnetic Resonance Spectroscopy, Molecular Sequence Data, N-Acetylglucosaminyltransferases, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Static Electricity
Amino Acid Sequence, Animals, Glycosylation, Humans, Magnetic Resonance Spectroscopy, Molecular Sequence Data, N-Acetylglucosaminyltransferases, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Static Electricity
J. Biol. Chem.
Date: Nov. 21, 2008
PubMed ID: 18809682
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