Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD.
Npl4 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, VCP(Ufd1-Npl4) 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 Npl4 deletion mutants we have revealed that the binding of Ufd1 to Npl4 is mediated by two regions: a conserved stretch of amino acids from 113 to 255 within the zf-Npl4 domain and by the Npl4 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 any 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 ERAD substrate alphaTCR. 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.
Lass
Agnieszka
A
Department of Anatomy and Cell Biology, Indiana University School of Medicine, 8600 University Boulevard, Evansville, IN 47712, USA.
McConnell
Elizabeth
E
Fleck
Krista
K
Palamarchuk
Alla
A
Wójcik
Cezary
C
D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs; D015415:Biomarkers / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017384:Sequence Deletion; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; D015415:Biomarkers; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; C514112:NPLOC4 protein, human; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C491497:UFD1 protein, human