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

ABSTRACT: 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.

SUBMITTER: Bailey CC

PROVIDER: S-EPMC3820858 | biostudies-other | 2013 Nov

REPOSITORIES: biostudies-other

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

Bailey Charles C CC Kondur Hema R HR Huang I-Chueh IC Farzan Michael M

The Journal of biological chemistry 20130925 45

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 m ...[more]

PMID: 24067232

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Project description:Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has alpha-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268. 2699-2704; Yang. Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382-386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the alpha-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have alpha-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-I cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (I) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) alpha-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the alpha-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulse chase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.

Dataset Information

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

Publications

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

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