Figure 1.
Sequence alignment of TMCC proteins.
(A) Human TMCC family members. (B) Domain structures of human TMCC proteins. (C) TMCC1 in various organisms. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, Xenopus laevis; Dr, Danio rerio; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans.
Figure 2.
Expression of TMCC1 protein in human cells.
(A) Whole cell extracts of HeLa cells were collected and immunoblotted with pre-immune serum or TMCC1 antibody. (B) HeLa cells were transfected with TMCC1 siRNA (siTMCC1-1 or siTMCC1-2) or a control siRNA; 72 h post-transfection, whole cell extracts were prepared and immunoblotted for TMCC1. α-Tubulin was stained as a control. (C) Whole cell extracts of various human cell lines were collected and immunoblotted for TMCC1; β-actin was stained as a control.
Figure 3.
Subcellular localization of TMCC1.
(A) Saponin-extracted COS-7 cells were fixed with methanol and stained with both Sec61α and TMCC1 antibodies; the boxed area shown is magnified. (B–C) HeLa cells were transfected with plasmids encoding GFP-tagged TMCC1 full-length protein, TMCC1(1–575), or TMCC1(571–653); 24 h post-transfection, cells with low and high levels of the exogenous proteins were fixed with methanol and stained with an anti-calnexin antibody. A magnified view of the boxed area in (B) is shown. (D) COS-7 cells were transfected with plasmids encoding GFP-tagged TMCC1(571–615) or TMCC1(615–653); 24 h post-transfection, cells were fixed with paraformaldehyde and then permeabilized with 0.2% Triton X-100 for 10 min at room temperature. Cells were then stained with an anti-calnexin antibody. Scale bars, 10 µm.
Figure 4.
(A) Workflow of ER isolation. HeLa cells were homogenized in 0.3 M sucrose. After 2 centrifugations, the P2 pellet was resuspended in 1.25 M sucrose and subjected to discontinuous sucrose-gradient centrifugation, and then the distinct layers at interfaces were collected. (B) Various fractions from (A) were collected and immunoblotted for ER, ribosomal, and mitochondrial proteins.
Figure 5.
(A–B) COS-7 cells were transfected with a plasmid encoding N-terminal (A) or C-terminal (B) GFP-tagged TMCC1; 24 h post-transfection, cells were fixed with paraformaldehyde and then permeabilized with either 40 µg/mL digitonin for 5 min on ice or 0.2% Triton X-100 for 10 min at room temperature. Cells were then co-stained with GFP and calnexin antibodies. Scale bars, 10 µm. (C) HeLa cells were treated with various combinations of digitonin and trypsin and then immunoblotted for TMCC1 and cathepsin D. (D) Two possible models of TMCC1 topology. Model (i) shows a transmembrane topology with 2 transmembrane domains, and Model (ii) shows an intramembrane topology with 2 intramembrane domains.
Figure 6.
Homo- or hetero-dimerization or oligomerization of TMCC proteins.
(A) HEK293T cells were co-transfected with plasmids encoding GFP-TMCC1 and FLAG-tagged TMCC1 fragments; 24 h post-transfection, cell lysates were collected for anti-FLAG immunoprecipitation to test for interactions between FLAG- and GFP-tagged proteins by performing western blotting. A schematic representation of the TMCC1 constructs is presented alongside the blots. Vector, pFLAG-CMV2 vector. FL, full-length TMCC1. (B) HEK293T cells were transfected with GFP-tagged TMCC1(571–653), TMCC2, or TMCC3 plasmids; 24 h post-transfection, cell lysates were prepared for TMCC1 immunoprecipitation to test for interaction between TMCC1 and exogenous proteins. TMCC1 and GFP-tagged proteins were analyzed by western blotting.
Figure 7.
Interaction of TMCC1 with ribosomal proteins.
(A) HEK293T cells were transfected with FLAG-tagged TMCC1 plasmid or the vector; 24 h post-transfection, cell lysates were prepared for anti-FLAG immunoprecipitation. Immunoprecipitated proteins were visualized on the protein gel by staining with Coomassie Brilliant Blue R-250. The protein bands marked in the figure were identified by mass spectrometry. Vector, pFLAG-CMV2 vector. (B) HeLa cell lysates were collected for TMCC1 immunoprecipitation, and samples were immunoblotted for TMCC1 and the ribosomal protein RPS6. (C) HEK293T cells were transfected with plasmids encoding FLAG-tagged TMCC1 full-length protein or fragments; 24 h post-transfection, cell lysates were collected for anti-FLAG immunoprecipitation. Ribosomal and FLAG-tagged proteins were analyzed by western blotting. Vector, pFLAG-CMV2 vector. FL, full-length TMCC1. (D) Ribosomes prepared from HeLa cells were incubated with purified GST or GST-TMCC1(101–350) protein and then pulled down using GSH-beads; ribosomal and GST-tagged proteins were analyzed by western blotting.