The N-terminus of yeast peptide: N-glycanase interacts with the DNA repair protein Rad23

• Published in: Biochemical and biophysical research communications Vol. 323; no. 1; pp. 149 - 155
• Main Authors: Biswas, Shyamasri, Katiyar, Samiksha, Li, Guangtao, Zhou, Xiaoke, Lennarz, William J., Schindelin, Hermann
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.10.2004
• Subjects: Amino Acid Sequence; Analytical ultracentrifugation; Chromatography; Cloning, Molecular; DNA Repair; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; ERAD; Glycoproteins - chemistry; Molecular Sequence Data; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - chemistry; Peptide: N-glycanase; Proteasome; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Quality control; Rad23; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Sequence Homology, Amino Acid; Surface Plasmon Resonance; Time Factors; Two-Hybrid System Techniques; Ultracentrifugation; ERAD; Peptide: N-glycanase; Rad23; Quality control; Surface plasmon resonance; Analytical ultracentrifugation; Proteasome
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2004.08.061

Yeast peptide: N-glycanase (PNGase) is involved in the proteasomal degradation of misfolded glycoproteins where it interacts with the DNA repair protein Rad23 as first detected in a yeast two-hybrid assay and subsequently confirmed by biochemical in vivo analyses. Limited proteolysis of PNGase with trypsin led to the removal of both an N-terminal and a C-terminal stretch. Based on these truncations the N-terminal region of yeast PNGase was identified as being responsible for binding to Rad23. Secondary structure predictions of this region suggest that it is composed of a single, solvent-exposed α-helix. The interaction between PNGase and Rad23 was studied using surface plasmon resonance revealing an equilibrium binding constant of ∼2.5 μM. The oligomeric nature of Rad23 was also investigated using sedimentation equilibrium analysis. Although Rad23 exists as a dimer in solution, the monomeric form of Rad23 associates with a PNGase monomer in a 1:1 stoichiometric ratio.