Table 1.
Golgi localization percentage of the wild type and mutant UBIAD1 proteins.
Figure 1.
UBIAD1 is accumulated in the juxta-nuclear area inside HEK293, T24 and PC-3 cells.
A, Bioinformatics analysis indicates that UBIAD1 is a putative eight transmembrane protein. B, Subcellular localization of UBIAD1 in HEK293, T24 and PC-3 cells are similar. Left, UBIAD1-EGFP; Middle, Ds Red-Golgi (Golgi marker); Right, Merge of left and middle; Upper panel: HEK293 cells; Middle panel: T24 cells; Lower panel: PC-3 cells; Arrows point to UBIAD1-EGFP, the Golgi and the colocalization of UBIAD1 and the Golgi, respectively. C, UBIAD1 was fused with c-Myc-epitope in three different locations (N terminus, amino acid 102 and C terminus). D, UBIAD1 is mainly localized inside the cytosol. a, plasmid Myc-UBIAD1 was transfected into the HEK293 cells; b, plasmid UBIAD1-102 Myc was transfected into the HEK293 cells; c, plasmid UBIAD1-Myc was transfected into the HEK293 cells. pcDNA3.1-BFP was used to mark the whole cell morphology. The HEK293 cells were stained with c-Myc antibody under permeabilized and non-permeabilized conditions, respectively. For permeabilization of the cells, fixed cells were treated with 0.5% Triton X-100 in PBS (Amresco Inc.) for 20 minutes. All experiments were repeated at least three times. Size bar represents 10 µm.
Figure 2.
UBIAD1 is localized on the ER (endoplasmic reticulum) and the Golgi, but not on the plasma membrane, of both HEK293 cells and T24 cells.
A, UBIAD1 is localized on the Golgi apparatus of HEK293 cells. HEK293 cells were transfected with plasmid UBIAD1-EGFP and plasmid Golgi-RFP (Golgi marker). The nuclei were stained with DAPI. a, UBIAD1-EGFP expressed in HEK293 cells; b, nuclei stained with DAPI; c, the merge of a and b; d, Golgi marker expressed in HEK293 cells; e, the merge of b and d; f, the merge of a, b and d. Arrows point to the UBIAD1-EGFP. B, UBIAD1 is localized on the Golgi of a single HEK293 cell; a, UBIAD1-EGFP expressed in HEK293 cell; Arrows point to the UBIAD1-EGFP. b and e, Ds Red-Golgi marker; Arrows point to the Golgi. c, the merge of a and b; The nuclei in a, b and c were stained with DAPI. d, The endogenous UBIAD1 was measured. HEK293 cell stained with UBIAD1 antibody and the FITC-conjugated secondary antibody; Arrow point to the endogenous UBIAD1. f, the merge of d and e. C, UBIAD1 is localized on the ER of a single HEK293 cell; a, UBIAD1-EGFP expressed in a single HEK293 cell; b and e, Ds Red-ER marker; c, the merge of a and b; The nuclei in a, b and c were stained with DAPI. d, The endogenous UBIAD1 was measured. HEK293 cell stained with UBIAD1 antibody and the FITC-conjugated secondary antibody; f, the merge of d and e. The arrangement of arrows is similar to those in figure 2B. D, UBIAD1 is not detected on the plasma membrane. a, UBIAD1-EGFP expressed in a single HEK293 cell; b, RFP-H-Ras (marker for plasma membrane and the Golgi); Arrow points to plasma membrane. c, the merge of a and b; d, The endogenous UBIAD1 was measured. HEK293 cell stained with UBIAD1 antibody and the R-PE-conjugated secondary antibody; e, Orail1-EGFP (marker for plasma membrane); Arrow points to plasma membrane. f, the merge of d and e. E, As in HEK293 cells, UBIAD1 is expressed on the ER and the Golgi, but not on the plasma membrane, in bladder carcinoma T24 cells. a, d and g, UBIAD1-EGFP expressed in T24 cells; Arrows point to UBIAD1-EGFP. b, e and h, markers for the Golgi (b, Ds Red-Golgi), ER (e, Ds Red-ER) and plasma membrane (h, BFP-H-Ras), respectively; Arrows point to the Golgi, the ER and the plasma membrane. c, Merge of a and b; f, Merge of d and e; i, Merge of g and h. All experiments were repeated at least three times. Size bar represents 10 µm.
Figure 3.
UBIAD1 is localized in the Golgi of L02 cells.
A, Proteins were extracted from L02 cells and the isolated Golgi apparatus. The relative intensities of UBIAD1, β1, 4-GALT (Golgi marker) and β-actin bands are shown in the bar graph. IOD (Information Object Definition) was used to specify grayscale images. L02, L02 cells; GF, Golgi fraction; Graph error bars represent standard deviation. Experiments were repeated three times. P<0.05 B, The Golgi apparatus were isolated from the L02 cells and stained with the Golgi-specific neutral red dye by supravital staining. Arrows point to the Golgi fraction. C, Image of the purified Golgi fraction was acquired using Scanning Electronic Microscopy (SEM, 25,000X). Blue arrow, tubular profile; red arrow, cisternae; yellow arrow, secretory vesicle The image of a typical cisternae sectioned tangentially was enlarged on the right [23]. D, UBIAD1-EGFP is accumulated in the Golgi followed by transfection of L02 cells with pUBIAD1-EGFP. Left, UBIAD1-EGFP; Middle, Ds Red-Golgi; Right, merge of UBIAD1-EGFP and Ds Red-Golgi.
Figure 4.
The Golgi retention signal of UBIAD1 resides within its N terminus.
A, Schematic representation of the secondary structure of UBIAD1 and the subclones of UBIAD1-EGFP; For example, 1-1a only contains the first 80 amino acids of UBIAD1. B, HEK293 cells were transfected with UBIAD1-EGFP subclones with N terminus deleted (1-2, 2-2, 3-2 and 4-2). C, HEK293 cells were transfected with UBIAD1-EGFP subclones with N terminus remaining (2-1, 3-1 and 4-1) and some of the transmembrane segments deleted. D, HEK293 cells were transfected with UBIAD1-EGFP subclones 1-1a and 1-1b, respectively. E, HEK293 cells were transfected with Orail1-EGFP and UBIAD1-N-Orail1-EGFP respectively. F, Bladder carcinoma T24 cells were transfected with UBIAD1-EGFP subclones 1-2, 2-1, and 1-1a, respectively. The subcellular localizations of these mutant proteins are similar in both HEK293 and T24 cells. All experiments were repeated at least three times. Size bar represents 10 µm.
Figure 5.
RPWS as the Golgi retention signal of UBIAD1.
A, A series of deletion clones were constructed for UBIAD1-EGFP (i.e., 20AA, Δ20, the first 20 amino acids of UBIAD1 N terminus was deleted); B, Multiple alignment of the N terminus of the UBIAD1 putative orthologs from ten different species (including Drosophila melanogaster, Anopheles gambiae str. PEST, Danio rerio, Gallus gallus, Canis lupus familiaris, Bos Taurus, Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus) using program Geneious. RPWS is conserved across all ten species; C, RPWS (amino acids 54 to 57) of the UBIAD1 N terminus was predicted to be the Golgi retention signal by Signal IP program. Arrows indicate the predicted Golgi retention signal. D, Deletion clones made in A were sequentially transfected into the HEK293 cells. Arrows point to the Golgi. WT, wild type UBIAD1-EGFP; Δ20, the first 20 amino acids of UBIAD1 N terminus was deleted.
Figure 6.
Arginine 54 (R54) as the most crucial amino acid in the UBIAD1 Golgi retention signal RPWS.
A, RPWS was sequentially mutated to AAAA, AAWS, APWS, RAWS, and RPAA and was expressed in the HEK293 cells. Arrows point to the Golgi. B, HEK293 cells were transfected with the wild type and mutant UBIAD1-EGFP proteins (Δ50, the first 50 amino acids deleted; Δ55, the first 55 amino acids deleted; AAAA, RPWS→AAAA, RPWS mutated to AAAA; APWS, RPWS→APWS and RPAA, RPWS→RPAA). The percentage of the Golgi localization of each protein is calculated from more than 100 cells (see Table 1 and the supplementary power point files Figure S1.ppt, Figure S2.ppt, Figure S3.ppt, Figure S4.ppt, Figure S5.ppt and Figure S6.ppt for details). Number represents Mean±SD. Experiments were repeated three times. Arrows point to the Golgi and the Golgi localized UBIAD1. C, Bladder carcinoma T24 cells were transfected with full length UBIAD1-EGFP and UBIAD1-EGFP mutant proteins (RPWS→AAAA and RPWS→APWS) D, The predicted 3D structure of the UBIAD1 N terminus by using Rosetta 3.1 (http://www.rosettacommons.org/manuals/archive/rosetta3.1_user_guide/) Size bar represents 10 µm.
Figure 7.
Intracellular trafficking of UBIAD1.
UBIAD1 was transported from the ER (endoplasmic reticulum) to the Golgi through anterograde trafficking. HEK293 cells were transfected with UBIAD1-EGFP. Fluorescent images were taken 4.5 hr, 5 hr, 5.5 hr, 6 hr, 7 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24 hr and 48 hr following the transfection. Arrows point to the Golgi. All experiments were repeated three times. Size bar represents 10 µm.
Figure 8.
UBIAD1 was strongly retained on the Golgi.
HEK293 cells were transfected with UBIAD1-EGFP. Following 20 hours of transfection, the HEK293 cells were treated with cycloheximide (100 ug/mL) for 5 hrs (a–c), 10 hrs (d–f) and 20 hrs (g–i), respectively. Nuclei of the HEK293 cells were stained with DAPI. Arrows point to the Golgi. All experiments were repeated three times. Size bar represents 10 µm.
Figure 9.
UBIAD1 might be targeted to the Golgi by COPII-mediated mechanism.
A, HEK293 cells were transfected with pUBIAD1-EGFP and co-transfected with Ds Red-ER (ER marker) and Ds Red-Golgi (Golgi marker), respectively. Following 24 hours of transfection, transfected HEK293 cells were treated with DMEM (1normal, as control) and brefeldin A (BFA, 5 ug/mL) for 0.5 hr (2), 1 hour (3) and 5 hours (4). Line arrow: ER; Arrow head: the Golgi. B, Substitution of Arginine 54 (R54) to lysine (K) does not affect the Golgi localization of UBIAD1. UBIAD1-EGFP was site-directed mutagenised by substituting the Arginine 54 (R54) to lysine. The mutant clone was transfected into the HEK293 and T24 cells. C, A model representing the putative interactions between RPWS of the UBIAD1 and the GTP of the Sar1 subunit of the COPII complex; Dotted lines represent the possible interactions. Experiments were repeated at least three times. Size bar represents 10 µm.
Figure 10.
Localization of UBIAD1 on the Golgi influences its tumor suppressing activity.
The bladder carcinoma T24 cells were treated with wild type and mutant (RPWS→AAAA) UBIAD1, followed by the staining with the ANNEXIN V-FITC/PI Apoptosis Assay Kit. Cell counts were performed with a flow cytometer. A, Normal, T24 cells stained with FITC and PI; B, Mock control, T24 cells treated with Lipofectamine 2000 (stained with FITC and PI 48 hr later); C, UBIAD1, T24 cells transfected with pcDNA3.1-UBIAD1 (stained with FITC and PI 48 hr later); D, UBIAD1(RPWS→AAAA), T24 cells transfected with (RPWS→AAAA) mutant of UBIAD1 with RPWS changed to AAAA (stained with FITC and PI 48hr later); E, Comparison of the apoptosis effects (B2) between wild type and (RPWS→AAAA) mutant UBIAD1 on T24 cells. Numbers were expressed as Mean±SD, which is represented as graphing error bar. Normal: 4.5±0.7%, MC: 11.9±0.5%, UBIAD1∶29.5±1.2%, UBIAD1 (RPWS→AAAA): 24.7±1.2%. *P = 0.0002<0.05, #P = 0.0006<0.05. Each experiment was repeated three times. B1: Mechanically damaged cells; B2: Late apoptosis cells; B3: Normal cells; B4: Early apoptosis cells; F, Western blot analysis of the amount of UBIAD1 protein in T24 cells. Normal, MC, UBIAD1 and UBIAD1 (RPWS→AAAA) are as above. In all of the above wild type and mutant UBIAD1 proteins, no GFP is attached.