Functional and Structural Analysis Reveals Dual Function on C-Terminal α Helix of Alg13 Protein

Abstract

As one of the most frequent and common post-translational modifications, protein asparagine N-glycosylation is initiated with the biosynthesis of a highly conserved dolichol-linked oligosaccharide (LLO) Glc3Man9GlcNAc2 on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. The second step of this process is catalyzed by a UDP-N-acetylglucosamine transferase that is comprised of two subunits. Alg13 and Alg14. The interaction between Alg13 and 14 is crucial for UDP-GlcNAc iransferase 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 bioinformalics and molecular biological methods, we have undertaken a functional and structural analysis of yeast Alg13 and Alg14 proteins to elucidate the mechanism of their interaction. Mutational studies have demonstrated that a short C-terminal α helix of Alg13 is required for interaction with Alg14 and enzyme activity. Furthermore. the highly conserved hydrophobic amino acids in this α helix are required for the membrane-associated localization of Alg13. 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 α helix. Co-immunoprecipitation assays have confirmed that the C-terminal three amino acids of Alg14 are required for maintaining the integrity of the Alg13/Alg14 complex and this depends on iheir hydrophobicity. Interestingly, a protein stability assay indicated that deletion or destruction of the C-terminal α helix stabilized an inactive free Alg13. indicating the importance of this structure in the degradation of cytosolic Alg13. These results demonstrate that: (1) the formation of this heterooligomeric complex is mediated by a short C-terminal α helix of Alg13 in cooperation with the last three amino acids of Alg14 and (2) the terminal α helix also funclions as a degradalion signal for controlling the protein level of free Alg13 in cytosol.