title: Artificial targeting of misfolded cytosolic proteins to endoplasmic reticulum as a mechanism for clearance.
abstract: 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.
authors.affiliation: Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702 USA.
authors.forename: Fen
authors.initials: F
authors.lastname: Liu
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 USA.
authors.forename: Deanna M
authors.initials: DM
authors.lastname: Koepp
authors.affiliation: Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702 USA.
authors.forename: Kylie J
authors.initials: KJ
authors.lastname: Walters
mesh_terms: D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D003600:Cytosol; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D007527:Isoenzymes; D007976:Leupeptins; D008565:Membrane Proteins; D011506:Proteins; D025801:Ubiquitin; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde