title: Requirements for mouse mammary tumor virus Rem signal peptide processing and function.
abstract: Mouse mammary tumor virus (MMTV) encodes a Rev-like protein, Rem, which is involved in the nuclear export and expression of viral RNA. Previous data have shown that all Rev-like functions are localized to the 98-amino-acid signal peptide (SP) at the N terminus of MMTV Rem or envelope proteins. MMTV-SP uses endoplasmic reticulum-associated degradation (ERAD) for protein trafficking. Rem cleavage by signal peptidase in the ER is necessary for MMTV-SP function in a reporter assay, but many requirements for trafficking are not known. To allow detection and localization of both MMTV-SP and the C-terminal cleavage product, we prepared plasmids expressing green fluorescent protein (GFP) tags. N-terminal Rem tagging led to protein accumulation relative to untagged Rem and allowed signal peptidase cleavage but reduced its specific activity. C-terminal tagging also led to Rem accumulation yet dramatically reduced cleavage, GFP fluorescence, and activity relative to N-terminally tagged Rem (GFPRem). Substitutions of an invariant leucine at position 71 between the known RNA-binding and nuclear export sequences interfered with GFPRem accumulation and activity but not cleavage. Similarly, deletion of 100 or 150 C-terminal amino acids from GFPRem dramatically reduced both Rem and MMTV-SP levels and function. Removal of the entire C terminus (203 amino acids) restored both protein levels and activity of MMTV-SP. Only C-terminal GFP tagging, and not other modifications, appeared to trap Rem in the ER membrane. Thus, Rem conformation in both the ER lumen and cytoplasm determines cleavage, retrotranslocation, and MMTV-SP function. These mutants further characterize intermediates in Rem trafficking and have implications for all proteins affected by ERAD.
authors.affiliation: Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Hyewon
authors.initials: H
authors.lastname: Byun
authors.forename: Nimita
authors.initials: N
authors.lastname: Halani
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Gou
authors.forename: Andrea K
authors.initials: AK
authors.lastname: Nash
authors.forename: Mary M
authors.initials: MM
authors.lastname: Lozano
authors.forename: Jaquelin P
authors.initials: JP
authors.lastname: Dudley
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000821:virology; D060746:Endoplasmic Reticulum-Associated Degradation; D006801:Humans; D008324:Mammary Tumor Virus, Mouse / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D012192:Retroviridae Infections / Q000378:metabolism / Q000821:virology*; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D021382:Protein Sorting Signals; D014759:Viral Envelope Proteins
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