Requirements for mouse mammary tumor virus Rem signal peptide processing and function

Area of Science:

• Molecular Biology
• Virology
• Cell Biology

Background:

• Mouse mammary tumor virus (MMTV) utilizes a Rev-like protein, Rem, for nuclear export and expression of viral RNA.
• The signal peptide (SP) of MMTV Rem or envelope proteins contains all Rev-like functions and interacts with endoplasmic reticulum-associated degradation (ERAD) for protein trafficking.
• Signal peptidase cleavage in the ER is crucial for MMTV-SP function, but trafficking requirements remain incompletely understood.

Purpose of the Study:

• To investigate the role of Rem protein conformation and trafficking in MMTV-SP function.
• To characterize intermediates in Rem trafficking using green fluorescent protein (GFP) tagged mutants.
• To elucidate the impact of modifications and deletions on Rem cleavage, retrotranslocation, and MMTV-SP activity.

Main Methods:

• Preparation of plasmids expressing N-terminal and C-terminal GFP-tagged Rem proteins.
• Analysis of Rem cleavage by signal peptidase and protein accumulation.
• Assessment of MMTV-SP activity using reporter assays.
• Introduction of specific amino acid substitutions and C-terminal deletions in GFPRem.

Main Results:

• N-terminal GFP tagging allowed cleavage but reduced Rem activity; C-terminal tagging dramatically reduced cleavage, activity, and GFP fluorescence.
• Substitution of leucine 71 and C-terminal deletions impaired GFPRem accumulation and activity, while full C-terminus removal restored protein levels and activity.
• C-terminal GFP tagging uniquely trapped Rem in the ER membrane, suggesting conformational dependence of Rem trafficking and function.

Conclusions:

• Rem protein conformation in the ER lumen and cytoplasm dictates signal peptidase cleavage, retrotranslocation, and MMTV-SP function.
• Specific mutations and truncations provide insights into Rem trafficking intermediates and ERAD pathways.
• Findings have implications for understanding the trafficking and function of proteins processed via ERAD.