Interferon-induced transmembrane protein 3 is a type II transmembrane protein.
The interferon-induced transmembrane (IFITM) proteins are a family of small membrane proteins that inhibit the cellular entry of several genera of viruses. These proteins had been predicted to adopt a two-pass, type III transmembrane topology with an intracellular loop, two transmembrane helices (TM1 and TM2), and extracellular N and C termini. Recent work, however, supports an intramembrane topology for the helices with cytosolic orientation of both termini. Here we determined the topology of murine Ifitm3. We found that the N terminus of Ifitm3 could be stained by antibodies at the cell surface but that this conformation was cell type-dependent and represented a minority of the total plasma membrane pool. In contrast, the C terminus was readily accessible to antibodies at the cell surface and extracellular C termini comprised most or all of those present at the plasma membrane. The addition of a C-terminal KDEL endoplasmic reticulum retention motif to Ifitm3 resulted in sequestration of Ifitm3 in the ER, demonstrating an ER-luminal orientation of the C terminus. C-terminal, but not N-terminal, epitope tags were also degraded within lysosomes, consistent with their luminal orientation. Furthermore, epitope-tagged Ifitm3 TM2 functioned as a signal anchor sequence when expressed in isolation. Collectively, our results demonstrate a type II transmembrane topology for Ifitm3 and will provide insight into its interaction with potential targets and cofactors.
From the Department of Infectious Diseases, The Scripps Research Institute, Jupiter, Florida 33458. Electronic address: cbailey@scripps.edu.
Charles C
CC
Bailey
From the Department of Infectious Diseases, The Scripps Research Institute, Jupiter, Florida 33458.
Hema R
HR
Kondur
the Department of Cell Biology and Neuroscience, College of Natural and Agricultural Science, University of California, Riverside, California 92521.
I-Chueh
IC
Huang
From the Department of Infectious Diseases, The Scripps Research Institute, Jupiter, Florida 33458.
Michael
M
Farzan
D020816:Amino Acid Motifs; D000818:Animals; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017434:Protein Structure, Tertiary
D008565:Membrane Proteins; C462181:fragilis protein, mouse
Flow Cytometry; Ifitm3; Interferon; Membrane Proteins; Microscopic Imaging; Virus Entry