The N-terminus of yeast peptide: N-glycanase interacts with the DNA repair protein Rad23.
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 alpha-helix. The interaction between PNGase and Rad23 was studied using surface plasmon resonance revealing an equilibrium binding constant of approximately 2.5 microM. 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.
Biswas
Shyamasri
S
Department of Biochemistry and Center for Structural Biology, Centers for Molecular Medicine, SUNY Stony Brook, Stony Brook, NY 11794-5115, USA.
Katiyar
Samiksha
S
Li
Guangtao
G
Zhou
Xiaoke
X
Lennarz
William J
WJ
Schindelin
Hermann
H
D000595:Amino Acid Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D014461:Ultracentrifugation
D004268:DNA-Binding Proteins; D006023:Glycoproteins; C490846:RAD23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase