The zinc metalloprotease ZMPSTE24 binds a distinct topological isoform of the tail-anchored protein IFITM3

Figures

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  Figure 1.. Schematic representation of IFITM3 and ZMPSTE24 in a membrane bilayer. IFITM3 has a C-terminal TM span, two N-terminal alpha-helices, AH1 (which is amphipathic) and AH2, and three conserved cysteines (C71, C72 and C105) that can undergo S-palmitoylation (red squiggly lines). ZMPSTE24 has seven TM spans and an HExxH…E zinc metalloprotease domain. The Zn2+ ion (pink) is coordinated by residues H335, H339 and E415 (blue). The catalytic glutamate E336 (yellow) activates a water molecule for peptide hydrolysis.

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  Figure 2.. The mutant protein ZMPSTE24E336A, but not other HExxH…E mutants, shows enhanced binding to IFITM3 and the bound species is hypo-palmitoylated. (A) The ZMPSTE24E336A mutant co-IPs IFITM3 more effectively than WT ZMPSTE24 or other active site mutants. Vector or the indicated Flag-tagged ZMPSTE24 variants were co-transfected with myc-tagged IFITM3 in HEK293 cells and subjected to co-IP with anti-Flag agarose, SDS-PAGE, and Western blotting with anti-Flag and anti-myc antibodies. Flag-ZMPSTE24 (red) and myc-IFITM3 (green) proteins are indicated in the IP and whole cell lysate (WCL). The white arrow (Lane 4) indicates faster migrating myc-IFITM3 species only observed when co-expressing ZMPSTE24E336A. The ZMPSTE24-IFITM3 interaction was quantified from three independent experiments, with the average and standard deviation of the mean shown (normalized to ZMPSTE24E336A). It should be noted that ZMPSTE24, whether tagged, or untagged always migrates as 2 bands. (B) The myc-IFITM3 species that co-IP’s with Flag-ZMPSTE24 E336A migrates more rapidly than the species which co-IP’s with Flag-IFITM3. Vector, Flag-ZMPSTE24, Flag-ZMPSTE24E336A, or Flag-IFITM3 were co-transfected with myc-tagged…

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  Figure 3.. IFITM3 Cys105 forms an intermolecular disulfide bond with ZMPSTE24E336A in vitro. (A) HEK293 cells transfected with Flag-ZMPSTE24 or Flag-ZMPSTE24E336A and myc-IFITM3 were subjected to co-IP with anti-Flag agarose beads. Immune complexes were resolved by non-reducing (left) or reducing (right) SDS-PAGE prior to Western blotting with anti-Flag and anti-myc antibodies. A higher molecular weight species (~65 kDa) containing both the myc and Flag signals is detected exclusively under non-reducing conditions. (B) The disulfide-linked species containing ZMPSTE24E336A and IFITM3 is disrupted by NEM in the lysis buffer. Cells transfected with Flag-ZMPSTE24E336A were induced with interferon β for 18 hrs prior to lysis with 1% Triton X-100 buffer without (−) or with (+) 0.1mM NEM. Proteins were immunoprecipitated with anti-Flag agarose beads, resolved by non-reducing SDS-PAGE and analyzed by Western blotting with anti-Flag and anti-IFITM3 antibodies. * denotes a non-specific background band. (C) HEK293 cells were transfected with vector, Flag-ZMPSTE24 or Flag-ZMPSTE24E336A with the indicated myc-tagged IFITM3 cysteine variants and subjected to co-IP followed by non-reducing SDS-PAGE and…

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  Figure 4.. ZMPSTE24-Cys359 is necessary and sufficient to form the intermolecular disulfide bond with IFITM3-Cys105. (A) X-ray crystal structure (pdb: 5SYT) (1) of ZMPSTE24. Cysteines (red balls), Zn2+ (purple ball), and Zn2+-coordinating residues H335, H339 and E415 (blue) are indicated. Approximate position of membranes separating cytoplasm and lumen is shown. (B) Cys359 in ZMPSTE24E336A is required for disulfide bonding to IFITM3. The indicated Flag-tagged ZMPSTE24 E336A Cys mutants were co-expressed with myc-IFITM3 (or myc-IFITM3C105A, Lane 9) and subjected to co-IP and non-reducing SDS-PAGE as described above. ZMPSTE24E336A lacking cysteines (“Cys-less”) and C359A bind IFITM3 but do not form the intermolecular disulfide species. (C) Working model for the orientation of IFITM3’s TM span in the absence or presence of ZMPSTE24E336A. Schematic of fully palmitoylated IFITM3 in the Ncyto-Cexo topology (left) and hypo-palmitoylated IFITM3 in the Ncyto-Ccyto topology bound to ZMPSTE24 (right). Positions of the cysteines in IFITM3 and ZMPSTE24E336A that oxidize during co-IP are indicated.

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  Figure 5.. A split fluorescence topology reporter corroborates the expected Ncyto-Cexo topology for IFITM3. (A) Schematic of the split fluorescence reporter system developed by Mckenna et al. (52). To determine the cellular location of the N- and C-termini of IFITM3, the indicated plasmids expressing BFP and IFITM3 tagged with the β11 strands of mCherry and GFP at the N- or C-termini were stably transfected into 293 Flp-In TRex cells expressing cytosolic GFP1–10 and ER-localized mCherry1–10. Only when the β11 peptide binds to its partner protein does fluorescence occur, indicating cytoplasmic (GFP+) or ER lumenal (mCherry+) localization. (B) Fluorescence microscopy and (C) flow cytometry of cells expressing N-β11-IFITM3 or IFITM3-β11-C proteins of a representative experiment confirm an Ncyto-Cexo topology under steady state conditions. Scale bar = 100 μm. In C, the fluorescence patterns of cells are indicated for each quadrant.

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  Figure 6.. The C-terminus of IFITM3 is cytoplasmic in ZMPSTE24E336A-expressing cells suggesting an inverted orientation for IFITM3’s TM span. (A-C) Topology of IFITM3 constructs in the absence (vector) or presence of ZMPSTE24E336A. Flp-In 293 TREx topology reporter cells expressing (A) N-β11-IFITM3, (B) IFITM3-β11-C or (C) IFITM3C71A-β11-C were transfected with vector control or ZMPSTE24E336A and analyzed by fluorescence microscopy. Scale bar = 100 μm. (D) Graph of cells in A-C were analyzed by flow cytometry (shown in Fig. S3). The mean GFP:BFP ratio was calculated for each condition, and the average and standard deviation fold change (n=6) compared to vector alone, set to 1, is shown. ** p<0.005, **** p<0.0005 in comparison to vector control (−).

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  Figure 7.. The C-terminus of IFITM3 bound to ZMPSTE24E336A was exposed to the ER lumen during its biogenesis. (A) Schematic of myc-tagged IFITM3 proteins with no opsin glycosylation tag, or an opsin tag at the N- or C-terminus. (B) Co-IP by Flag-tagged ZMPSTE24E336A of myc-IFITM3 proteins with N- and C-terminal opsin tags. Immune complexes were mock-treated (−) or treated with PNGase F (+) prior to non-reducing SDS-PAGE and Western blotting. The opsin tag at the C-terminus of IFITM3 is glycosylated and shifts upon PNGase treatment, as is also the case for the ~65kDa intermolecular disulfide complex (absent when using ZMPSTE24E336A, C359A, lanes 7 and 8). A dotted line was added to highlight protein gel mobility differences.

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  Figure 8.. The IFITM3 Ncyto-Ccyto topological isoform is unstable and degraded in a ubiquitin/proteasome-dependent manner. (A) Flow cytometry histogram of Flp-In 293 TREx cells expressing IFITM3-β11-C analyzed after 4 hrs of treatment with vehicle (DMSO) or inhibitors of the E1 ubiquitin-activating enzyme (E1 inhib), proteasome (Btz), p97/VCP (p97 inhib), or the protein synthesis inhibitor cycloheximide (CXH) in the absence or presence of p97 inhibitor. The GFP:BFP ratio increases upon inhibition of the ubiquitin-proteasome system. (B) Cells expressing N-β11-IFITM3, IFITM3-β11-C, or ASGR1-β11-C were treated with the indicated drugs for 4 hrs prior to analysis by flow cytometry. The average and standard deviation of the fold change GFP:BFP ratio over DMSO vehicle is shown (n=4). * p<0.05, **** p<0.0005 in comparison to DMSO treatment (−).

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  Figure 9.. Model for ZMPSTE24’s potential role(s) in the quality control of IFITM3 topology. IFITM3 is initially inserted into the ER membrane in the Ncyto-Cexo topology. Under-palmitoylation of IFITM3 may cause the TM span to flip in the membrane, possibly mediated by ZMPSTE24, resulting in the Ncyto-Ccyto topology. ZMPSTE24 may instead assist in correction of the TM span of IFITM3, allowing trafficking to the Golgi and ultimately the plasma membrane or endosomes where it functions as a viral restriction factor. If not rectified, the Ncyto-Ccyto IFITM3 altered topological form undergoes ubiquitination, membrane extraction and degradation by the proteasome (ERAD). The ZMPSTE24E336A mutant may trap the normally transient inverted form of IFITM3 shown in the middle.