Interferon-induced transmembrane protein 3 is a type II transmembrane protein

Area of Science:

• Virology
• Cell Biology
• Structural Biology

Background:

• Interferon-induced transmembrane (IFITM) proteins are key regulators of viral entry into host cells.
• Previous models proposed a type III transmembrane topology for IFITM proteins, but recent evidence suggests alternative orientations.

Purpose of the Study:

• To determine the precise transmembrane topology of murine Ifitm3, a critical antiviral IFITM protein.
• To elucidate the orientation of Ifitm3's N and C termini within the cell membrane.

Main Methods:

• Antibody-based cell surface staining to assess N and C terminus accessibility.
• Functional analysis of a C-terminal KDEL ER retention motif to probe C-terminal orientation.
• Lysosomal degradation assays for epitope-tagged Ifitm3 variants.
• Expression of Ifitm3 transmembrane domain 2 (TM2) as a signal anchor sequence.

Main Results:

• The N terminus of Ifitm3 showed cell type-dependent cell surface accessibility, indicating a minor pool with extracellular orientation.
• The C terminus was predominantly accessible at the cell surface, suggesting a major extracellular orientation.
• KDEL motif addition led to ER sequestration, confirming C-terminal ER-luminal orientation.
• Lysosomal degradation of C-terminal tags, but not N-terminal tags, supported luminal orientation.
• Ifitm3 TM2 functioned as a signal anchor sequence.

Conclusions:

• Murine Ifitm3 adopts a type II transmembrane topology, with the C terminus oriented extracellularly.
• This topology provides crucial insights into Ifitm3's mechanism of action, including interactions with viral targets and cellular cofactors.