We report that misfolded cytosolic proteins can be cleared from mammalian cells by directing them to endoplasmic reticulum (ER). NAT1 R64W and Parkin R42P are naturally occurring misfolded variants of cytosolic enzymes that acetylate arylamines and ubiquitinate proteins, respectively. We demonstrate that proteasome inhibition causes ER accumulation of NAT1 R64W and its ubiquitinated species, and that these products are cleared from cells following inhibition release. NAT1 WT by contrast is stable and not present at ER. The R42P mutation in Parkin locates to a UBL domain that interacts with C-terminal domains. Parkin R42P full length protein is trafficked poorly to ER and stable. Interestingly, fusion of the isolated R42P UBL to NAT1 WT results in a fusion product that is trafficked robustly to ER and degraded. Thus, the misfolded UBL is apparently masked by the intramolecular interactions. We also find that artificially directing Parkin R42P to ER by fusion with the Sec61β ER-directing transmembrane domain triggers its clearance. Altogether, our results suggest that routing misfolded cytosolic proteins to ER may be an effective strategy for clearance.
Bibliographical noteFunding Information:
We are indebted to Patrick Hanna (University of Minnesota) for useful discussions, Gia Voeltz (University of Colorado) for mCherry-Sec61β, BFP-Sec61β and mCherry-Rab7 plasmids, Poul H. Jensen (University of Aarhus, Denmark) for SHSY5Y cells stably expressing Parkin WT or R42P, Ted Dawson (The Johns Hopkins University School of Medicine) for Myc-Parkin R42P plasmid, Jadranka Loncarek (NCI) for technical advice and sharing her microscope, Vinay Pathak and R. Andy Byrd (NCI) for sharing instruments, Stephen Lockett and Valentin Magidson at the Optical Microscopy and Analysis Laboratory (NCI) for technical assistance. This research was funded by grants from the NIH (CA117888 to KJW, GM076663 to DMK), American Cancer Society (RSG-07-186-01-GMC to KJW), and by the Intramural Research Program of the NIH, NCI, CCR.