Abstract
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.
Highlights
Protein asparagine N-glycosylation is one of the most frequent and common protein modifications that is important because it required for the structure and function of glycoproteins
Stereo Structure Suggests the Importance of Termini for Formation of the Alg13/Alg14 Complex—Primary sequence alignment only shows a low similarity between yeast Alg13, Alg14 proteins, and E. coli MurG
The assembly of the Alg13 and Alg14 subunits is critical for the activity of the UDP-N-acetylglucosamine transferase and contributes to the regulation of N-linked glycosylation in eukaryotic cells
Summary
Homology Modeling of the Alg13/Alg Complex—A refined three-dimensional homology model of the yeast Alg13/14 complex was first constructed based on the crystal structure of E. coli MurG (PDB code: 1NLM Chain A) using the Modeler 9.1 [16, 17]. (iii) To further optimize a large loop in yeast Alg subunit (from 53 to 79 residues), the best model selected in the second step was again applied to Loopmodel class. The DOPE scores calculated for the model (yeast Alg13/14 complex) and MurG were Ϫ38927.46 and Ϫ41430.95, respectively. To reconstruct the model of the Alg13/14 complex, orientation of last 12 amino acids (from Ser191 to Ser-202) including the C-terminal ␣-helix in 2jzc was re-directed based on the structure information of MurG. The minimization for this re-orientation was conducted by using MOE (Chemical Computing Group Inc.) with Amber force fields. To detect the FLAG-tagged proteins, the membrane was blotted with a rabbit anti-FLAG polyclonal antibody (Rockland, Inc.) followed by a secondary anti-rabbit antibody conjugated to alkaline phosphatase (Chemicom, International)
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