Figure 5.

NMNAT2 is polyubiquitinated by PAM and degraded by the proteasome. A, coIP from transfected 293 cells showing FLAG–FBXO45 increases binding of NMNAT2–MYC to GFP–PAM D5. B, PAM strongly stimulates polyubiquitination of NMNAT2 when the proteasome is inhibited with MG132. C, NMNAT2–MYC protein turnover visualized using the translation inhibitor cycloheximide (CHX). NMNAT2 turnover is reduced by MG132 inhibition of the proteasome. D, NMNAT2 turnover is reduced in two PAM knockout cell lines generated by CRISPR. E, NMNAT2 protein turnover is faster in the presence of WT PAM than catalytically inactive PAM LD. F, summary showing PAM/FBXO45/SKP1 complex polyubiquitinating NMNAT2. A–C and E, representative is shown of at least three independent experiments. D, shown is a representative of two independent experiments, in which PAM knockout cell lines were derived de novo each time using two different PAM gRNAs. IP, immunoprecipitation.