Analysis of Np14 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Np14 in ERAD

Np14 is a 67 kDa protein forming a stable heterodimer with Ufd1, which in turn binds the ubiquitous p97/VCP ATPase. According to a widely accepted model, VCPUdf1-Np14 promotes the retrotranslocation of emerging ER proteins, their ubiquitination by associated ligases, and handling to the 26S proteasome for degradation in a process known as ERAD (ER-associated degradation). Using a series of Np14 deletion mutants we have revealed that the binding of Ufd1 to Np14 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Np14 domain and by the Np14 homology domain between amino acids 263 and 344. Within the first region, we have identified two discrete subdomains: one involved in Ufd1 binding and one regulating VCP binding. Expression of ally one of the mutants failed to induce any changes in the morphology of the ER or Golgi compartments. Moreover, we have observed that overexpression of all the analyzed mutants induced mild ER stress, as evidenced by increased Grp74/BiP expression without associated XBP1 splicing or induction of apoptosis. Surprisingly, we have not observed any accumulation of the typical EPAD substrate alpha TC. This favors the model where the Ufd1-Npl4 dimer forms a regulatory gate at the exit from the retrotranslocone, rather than actively promoting retrotranslocation like the p97VCP ATPase. (C) 2008 Elsevier Inc. All rights reserved,