Fig 1.
Engineering the UAA system and demonstrating efficient incorporation of Azi in T. gondii.
(A) Diagram showing the use of amber stop codon suppression to incorporate UAAs into a nascent peptide strand using the endogenous translation machinery. (B) Chemical structure of the photoreactive UAA p-azidophenylalanine (Azi). Exposure of Azi to UV-A (365-nm) ultraviolet light causes the azide group to irreversibly form a reactive nitrene intermediate, which forms covalent crosslinks with proximal proteins. (C) Construct showing the Ty1-tagged aminoacyl-tRNA synthetase (E2AziRS) driven by the constitutively active GRA1 promoter and three tandem cassettes of the cognate amber suppressor tRNA driven by the RNA polymerase III–specific U6 promoter. (D) IFA showing that stably expressed E2AziRS localizes to the parasite cytoplasm as expected. Green, mouse anti-Ty1. Scale bar represents 2 μm. (E) Construct showing the SAG1 gene driven by the GRA1 promoter, in which the second amino acid F2 has been mutated to an amber codon. (F) IFA showing RHΔhxgprtΔsag1 parasites stably transfected with the synthetase/tRNA and SAG1 constructs. E2AziRS expression is confirmed by anti-Ty1 staining. Without Azi in the growth medium, SAG1 is not detected due to the in-frame stop codon. Upon addition of Azi, robust expression of SAG1 is observed trafficking properly to the cell periphery. Red, rabbit anti-SAG1 antibody; green, mouse anti-Ty1 antibody. Scale bar represents 2 μm. (G) Western blot of whole cell lysates shows expression of the SAG1 nonsense mutant only when Azi is added to the medium. Wild-type RH parasites expressing endogenous SAG1 are used as a control. Azi, p-azidophenylalanine; E2AziRS, Azi-tRNA synthetase; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyl transferase; IFA, immunofluorescence assay; SAG1, surface antigen 1; UAA, unnatural amino acid; WT, wild-type.
Fig 2.
Site-specific crosslinking of UPRT in T. gondii.
(A) The UPRT homodimer is stabilized by a β-arm (darker pink, structure adapted from PDB entry 1BD4). L92 and Y96 were chosen for Azi substitution based on orientation towards the other subunit in the crystal structure. (B) IFA showing cytoplasmic localization for parasites expressing the Y96 mutant UPRT-HA upon addition of Azi. Red, mouse anti-HA. Scale bar represents 2 μm. (C) Schematic for UPRT dimer formation using a second copy of UPRT with a Myc tag. UPRT proteins will assemble as either HA/HA or Myc/Myc homodimers, or a HA/Myc heterodimer. As the Myc-tagged monomers lack Azi, they should be crosslinked only when bound to an Azi-containing UPRT-HA partner. (D) Anti-HA western blot of UPRT crosslinking using strains expressing the synthetase/tRNA and either WT, L92, or Y96 UPRT-HA. Uncrosslinked UPRT migrates at 27 kDa (blue arrow). A small amount of UPRT is observed without Azi, indicating that nonspecific incorporation of other amino acids can occur, but this material is low abundance and lacks crosslinking ability. In the +Azi/+UV conditions, shifted products can be observed for both L92 and Y96 lines (red arrowheads), indicating successful crosslinking of a UPRT dimer. (E) Immunoblot of the UPRT crosslink samples with anti-Myc antibody verifies that the shifted products are covalently crosslinked UPRT homodimers. The Myc blot shows a lower relative efficiency of crosslinked to uncrosslinked material, presumably because the Myc-tagged monomers can only crosslink as the heterodimer, while the HA-tagged UPRT can crosslink as both the heterodimer and an HA/HA homodimer. Azi, p-azidophenylalanine; HA, hemagglutinin; IB, immunoblot; IFA, immunofluorescence assay; PDB, Protein Data Bank; UPRT, uracil phosphoribosyltransferase; WT, wild-type.
Fig 3.
Site-specific crosslinking of ILP1 reveals multiple potential binding partners.
(A) Diagram of ILP1 showing an N-terminal putative EF-hand domain (gray) followed by a coiled-coil domain (yellow). Also shown is the JPred secondary structure prediction of ILP1 revealing alpha-helices (black bars) and beta-strands (white arrows), as well as buried residue prediction used for choosing likely exposed residues for amber substitution [34]. Also noted are the potentially myristoylated glycine at position two (teal) and tandem cysteine prenylation/palmitoylation motifs internally and at the extreme C terminus (purple). Fourteen residues in ILP1 were chosen for amber mutagenesis to test for Azi-mediated crosslinking (green). (B) COILS prediction of ILP1; the window size refers to the number of residues used in the analysis. (C) Representative IFA of the Q168 ILP1 mutant containing Azi, which localizes properly to the parasite periphery. Red, rabbit anti-HA antibody; green, mouse anti-Ty1 antibody. Scale bar represents 2 μm. (D) Western blot of the ILP1 Azi mutants after UV irradiation reveals three major crosslinked species (red arrowheads). A smaller upshift (approximately 65 kDa) is observed for residues Y160 and Q168, with weak similar products for T152 and Q170. Residues T184, T187, and I188 exhibit a major band at approximately 200 kDa. E209 and K212 form a third upshift at approximately 140 kDa. Uncrosslinked ILP1 is denoted by the blue arrows (approximately 35 kDa). Azi, p-azidophenyalanine; E2AziRS, Azi-tRNA synthetase; HA, hemagglutinin; IB, immunoblot; IFA, immunofluorescence assay; ILP1, IMC localizing protein 1.
Fig 4.
IMC3 is a direct binding partner of ILP1.
(A) ILP1-3xHA amber mutants yielding the two large upshifted bands (T184, T187, I188, and E209) were expressed in an endogenously tagged IMC3-3xMyc background that also contains the synthetase/tRNA pair. Following Azi addition and UV irradiation, the uncrosslinked (blue arrow) and crosslinked species (black asterisks) can be reproduced in this strain. When probing for IMC3-3xMyc, uncrosslinked protein is observed (blue arrow), but a persistent high molecular weight background prevents confirmation of any potential upshift of IMC3. (B) Strategy for denaturing IP. Boiling in SDS disrupts the parasite’s cytoskeleton and protein–protein interactions of its components. The lysate is diluted to RIPA conditions for IP, and only the target protein (red) and covalently attached partners (green) are purified. (C) Western blot analysis of ILP1 denaturing IP recapitulates uncrosslinked (blue arrow) and crosslinked (black asterisks) ILP1 Azi mutants. As seen in the anti-Myc blot, the IP procedure eliminates the majority of the higher molecular weight background and reveals Myc-reactive crosslinked bands for residues T184, T187, and I188 (red arrowhead), demonstrating that IMC3 is indeed the partner at these residues. A small amount of uncrosslinked IMC3 that is likely to be reassociating with ILP1 is seen following dilution of the denatured lysate for IP (blue arrow). In contrast, no anti-Myc signal is seen for E209, indicating that this residue does not bind to IMC3. Azi, p-azidophenylalanine; HA, hemagglutinin; ILP1, IMC localizing protein 1; IP, immunoprecipitation.
Fig 5.
The C-terminal region of IMC3 is required for binding to ILP1 at T187.
(A) Diagram and IFA of IMC3 truncations expressed in Toxoplasma to determine which region of IMC3 binds to ILP1 T187. In endogenously tagged IMC3-3xMyc parasites, regions corresponding to alveolin only (112–279, IMC3A) and alveolin plus C terminus (112–538, IMC3AC) were tagged with V5 and localized. IMC3A partially mislocalizes to the maternal cytoplasm, suggesting that although the alveolin domain plays a role in IMC targeting, the inclusion of the C-terminal region of the protein improves IMC targeting similarly to wild-type IMC3. Red, mouse anti-V5 antibody; green, rabbit anti-Myc antibody. Scale bar represents 2 μm. (B) Western blot showing the high molecular weight product corresponding to a crosslinked full-length IMC3 in parasites expressing either IMC3A or IMC3AC (black asterisk). However, another smaller band (red arrowhead) is seen in the IMC3AC lysate, likely representing an ILP1/IMC3AC crosslinked product. The uncrosslinked ILP1 Azi mutant is denoted with a blue arrow. (C) ILP1-3xHA T187 denaturing IP shows the same ILP1 shifted products seen in whole cell lysates (first panel, black asterisk and red arrowhead), but an anti-V5 blot now clearly labels a band migrating at the same position as the new smaller anti-HA upshifted product (second panel, red arrowhead), demonstrating that this species corresponds to ILP1 T187 crosslinked to the IMC3AC. The light chain (lc) signal seen at 25 kDa obscures detection of residual uncrosslinked IMC3A. However, detecting the original ILP1 T187/IMC3 full length species in the anti-Myc blot (third panel, black asterisk) shows that IP in both IMC3A and IMC3AC conditions was successful. Uncrosslinked proteins are denoted with blue arrows. (D) Western blot analysis of anti-V5 denaturing IP performed with the same strains. Both IMC3A and IMC3AC are robustly enriched (second panel, blue arrows), but crosslinked ILP1 is only obtained in the IMC3AC condition (first panel, red arrowhead). Some uncrosslinked IMC3-3xMyc is seen in the IP, likely reflecting interactions of this abundant alveolin in the lysate (third panel). Uncrosslinked proteins are denoted with blue arrows, and both heavy chains (hc) and lc are seen as extra signal in the anti-V5 blot. A, alveolin domain only; AC, alveolin domain and C-terminus; Azi, p-azidophenylalanine; HA, hemagglutinin; hc, heavy chain; IFA, immunofluorescence assay; ILP1, IMC localizing protein 1; IP, immunoprecipitation; lc, light chain; trunc., truncation.
Fig 6.
IMC6 is another direct binding partner of ILP1.
(A) Crosslinking of T187 and E209 in strains endogenously 3xMyc-tagged for IMC3, IMC4 or IMC6. Tagging of IMC6 results in slower migration for the E209 Azi crosslinked product compared to the IMC3/4-3xMyc lines, which can be attributed to the addition of the epitope tag. A subtle shift can also be seen in the T187/ IMC3-3xMyc strain (black asterisk), as IMC3 is the partner at this residue. Uncrosslinked ILP1 Azi mutants are denoted with blue arrows. (B) Denaturing ILP1 co-IP to verify the E209/IMC6 interaction using IMC6 tagged parasites (T187, which binds IMC3, is used as a control). The anti-HA blot shows the expected uncrosslinked material (first panel, blue arrow) and crosslinked products (first panel, black asterisks) in the tagged lines, but only the E209 product is detected with anti-Myc, confirming the interaction with IMC6 (second panel, red arrowhead). Uncrosslinked IMC6, which was mostly removed by denaturation, is also present in both conditions (second panel, blue arrow). Azi, p-azidophenylalanine; co-IP, co-immunoprecipitation; HA, hemagglutinin; ILP1, IMC localizing protein 1; IMC, inner membrane complex.
Fig 7.
The N-terminal region of IMC6 is required for binding to ILP1 at E209.
The experimental design mimics the one reported for IMC3 in Fig 6. (A) Alveolin only (128–290, IMC6A) and alveolin plus C terminus (128–444, IMC6AC) truncations tagged with a V5 epitope were visualized. Both truncations localize to the growing daughter IMC but also exhibit partial mislocalization in the maternal cytoplasm, which does not appear to be rescued with the addition of the C-terminal region. Red, mouse anti-V5 antibody; green, rabbit anti-Myc antibody. Scale bar represents 2 μm. (B) Western blot of whole cell lysate showing uncrosslinked ILP1 (blue arrow) and the original upshifted species (black asterisk) but no visible lower molecular weight band that would indicate crosslinking to the smaller size truncations. (C) ILP1-3xHA E209 denaturing IP enriches for ILP1 (first panel), but no upshift is observed in the anti-V5 blot (second panel), indicating that neither IMC6A nor IMC6AC is sufficient to crosslink to ILP1 at residue E209. The IMC6A band is obscured by antibody light chain (lc) cross-reactivity. The anti-Myc blot (third panel) confirms successful IP, as seen by the original ILP1 E209/IMC6 full-length upshift (black asterisk). Uncrosslinked proteins are denoted with blue arrows. (D) Anti-V5 denaturing IP enriching for the IMC6 truncations correspondingly lacks any ILP1-3xHA signal (first panel). Uncrosslinked proteins are denoted with blue arrows, and both heavy chains (hc) and lc are seen as extra signal in the anti-V5 blot. (E) An N terminus plus alveolin domain truncation of IMC6 (1–290, IMC6NA) appears to rescue localization like wild-type IMC6. Red, mouse anti-V5 antibody; green, rabbit anti-Myc antibody. (F) Anti-HA immunoblot of both whole cell lysate (first panel, red arrowhead) and after anti-HA denaturing IP (second panel, red arrowhead) reveals a smaller upshifted species (approximately 90 kDa) for the IMC6NA strain, suggesting that ILP1 E209 is crosslinking to this mutant. Anti-V5 blot confirms that IMC6NA is detected at this molecular weight size (third panel, red arrowhead), demonstrating the identity of an ILP1 E209/IMC6NA crosslink. Anti-Myc blot confirms successful co-IP of the original IMC6 upshift (fourth panel). Uncrosslinked ILP1 is denoted with blue arrows, and the original upshift corresponding to ILP1 crosslinked to full-length IMC6 is denoted with black asterisks. A, alveolin domain only; AC, alveolin domain and C-terminus; Azi, p-azidophenylalanine; co-IP, co-immunoprecipitation; HA, hemagglutinin; hc, heavy chain; ILP1, IMC localizing protein 1; IMC, inner membrane complex; IP, immunoprecipitation; lc, light chain; NA, N-terminus and alveolin domain; wt, wild-type.
Fig 8.
Mass spectrometric identification of crosslinked proteins reveals IMC27 as an ILP1 binding partner.
(A) Western blot (W) and Coomassie gel (C) analyses of large-scale denaturing IP of Y160. The region of gel containing the crosslinked ILP1 species (red arrowhead) was excised and processed for LC-MS/MS peptide identification. Uncrosslinked ILP1 is denoted with a blue arrow. (B) Detergent fractionation showing that IMC27 is firmly associated with the IMC cytoskeleton, like ILP1 [17]. IMC1 is a control for the insoluble fraction whereas membrane-associated ISP3 is readily solubilizes upon detergent extraction. (C) Photocrosslinking of Y160 and Q168 in strains endogenously 3xMyc tagged for IMC27 (or IMC12 as a control). Tagging of IMC27 results in slower migration of the crosslinked product compared to that of the IMC12 tagged strain, indicating IMC27 is the partner. The shifted products are also detected in the anti-Myc blot (second panel, red arrowhead). Uncrosslinked proteins are denoted with blue arrows. (D) Denaturing IP shows the same pattern of shifted products for ILP1. Probing the samples with anti-Myc (for IMC29 or IMC12) confirms the higher migrating shifted product are IMC27 (red arrowhead), while uncrosslinked IMC27 is largely removed (blue arrow). IMC12-3xMyc is completely eliminated by denaturation. Uncrosslinked proteins are denoted with blue arrows. (E) IFA of IMC27-3xMyc parasites reveals that it localizes solely to the maternal IMC, indicating that interaction with ILP1 via Y160 and Q168 occurs within this subcompartment rather than the forming daughters. Red: mouse anti-Myc antibody, green: rat anti-ILP1 antibody. Scale bar represents 2 μm. Azi, p-azidophenylalanine; HA, hemagglutinin; IFA, immunofluorescence assay; ILP1, IMC localizing protein 1; IMC, inner membrane complex; IP, immunoprecipitation; ISP3, IMC subcompartment protein 3; LC-MS/MS, liquid chromatography tandem mass spectrometry.