title: Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation.
abstract: Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation.IMPORTANCEBacterial and viral infections produce pathogen-specific proteins that interfere with host functions, including the immune response. Mouse mammary tumor virus (MMTV) is a model system for studies of human complex retroviruses, such as HIV-1, as well as cancer induction. We have shown that MMTV encodes a regulatory protein, Rem, which is cleaved into an N-terminal signal peptide (SP) and a C-terminal protein (Rem-CT) within the endoplasmic reticulum (ER) membrane. SP function requires ER membrane extraction by retrotranslocation, which is part of a protein quality control system known as ER-associated degradation (ERAD) that is essential to cellular health. Through poorly understood mechanisms, certain pathogen-derived proteins are retrotranslocated but not degraded. We demonstrate here that MMTV SP retrotranslocation from the ER membrane avoids degradation through a unique process involving interaction with cellular p97 ATPase and failure to acquire cellular proteasome-targeting sequences.
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Hyewon
authors.initials: H
authors.lastname: Byun
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Poulami
authors.initials: P
authors.lastname: Das
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Houqing
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Aleman
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Mary M
authors.initials: MM
authors.lastname: Lozano
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Matouschek
authors.affiliation: Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA jdudley@austin.utexas.edu.|Center for Infectious Disease, The University of Texas at Austin, Austin, Texas, USA.|Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Jaquelin P
authors.initials: JP
authors.lastname: Dudley
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D057131:Immune Evasion; D008324:Mammary Tumor Virus, Mouse / Q000276:immunology* / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D059748:Proteolysis; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; D014764:Viral Proteins; C530424:derlin-1 protein, mouse; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C404942:p97 ATPase
keywords: ERAD; retrotranslocation; retrovirus; signal peptide