VCP/p97 AAA-ATPase does not interact with the endogenous wild-type cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, DeltaF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify DeltaF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds DeltaF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR-VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.
Goldstein
Rebecca F
RF
Department of Cell Biology, Comprehensive Cancer Center, and Gregory Fleming James Cystic Fibrosis Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
Niraj
Ashutosh
A
Sanderson
Todd P
TP
Wilson
Landon S
LS
Rab
Andras
A
Kim
Helen
H
Bebok
Zsuzsa
Z
Collawn
James F
JF
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D025821:Polyubiquitin / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020543:Proteome; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D020543:Proteome; D025821:Polyubiquitin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein