Role of N-Terminal region of zPKR1 for zMRAP2a and hMRAP2 interaction. (A) Alignments of the hPKR1, hPKR2, mPKR1 and zPKR1 N-terminal sequence. In red the amino acids that are conserved in zebrafish and mouse with respect to hPKR1 and hPKR2, in bold the amino acid that was replaced by photoreactive p-benzoyl-L-phenilalanine. (B) GST pull-down experiments. The zPKR1-NT-GST and hPKR1-NT-GST were used to pull down CT-domains of zebrafish zMRAP2a and of hMRAP2. The eluate obtained using zPKR1-NT-GST (ENTz) and eluate obtained using hPKR1-NT-GST(ENTh) were analysed by Western blotting analysis with anti-His antibody. The negative control (EGST) was obtained using GST to pull down zMRAP2a. (C) Schematic representation of the amber codon suppression technology for genetic introduction of the photoreactive p-benzoyl-L-phenilalanine (pBpa) directly into expressed zPKR1 in a yeast cell system. (D) Crosslinking of zPKR1-WT and the zPKR1-W11Bpa with zCT-MRAP2a domain. Membranes prepared from P. pastoris cells expressing the zPKR1-W11Bpa and zPKR1-WT were incubated with zCT-MRAP2a domain. The membrane proteins were immunoblotted and analysed with anti-His antibodies.
|
