Figure 1.
Expression of APH0859 in Anaplasma phagocytophilum and new annotation of aph0859 ORF.
A. Truncated rAts-1 (derived from predicted APH0859 ORF) expressed in E. coli was purified by immobilized Ni2+ affinity chromatography, and subjected to SDS-PAGE analysis followed by Coomassie Brilliant blue staining. Lanes: M, protein molecular mass marker; rAts-1, recombinant truncated A. phagocytophilum Ats-1. Arrow indicates rAts-1 migration. B. Western blot analysis of A. phagocytophilum (Ap)-infected and uninfected HL-60 cells using affinity-purified rabbit anti-Ats-1 antibody. Arrows 1 and 2 indicate full-length native Ats-1 and cleaved Ats-1, respectively. Molecular mass markers are indicated at the left. C. The amino acid sequence deduced from the newly defined aph0859 (ats-1) ORF. The amino acid sequences identified by mass spectrometry are highlighted in bold. The N-terminal mitochondrial targeting sequence predicted by the Mitoprot program is indicated in shaded bold. * indicates the stop codon.
Figure 2.
Translocation of Ats-1 from Anaplasma phagocytophilum into the cytoplasm of HL-60 cells.
A. Double immunofluorescence labeling of Ats-1 and P44 in A. phagocytophilum–infected HL-60 cells. Uninfected HL-60 cells and A. phagocytophilum-infected HL-60 cells at 22, 32, or 42 h post-infection were double immunofluorescence-labeled using mouse monoclonal anti-P44 (P44; Alexa Fluor 488, green) and affinity-purified rabbit anti-Ats-1 (Ats-1; Alexa Fluor 555, red), and subjected to epifluorescence microscopy (22 or 32 h p.i.) or to confocal laser scanning microscopy (42 h p.i.). Merged images with or without phase contrast micrograph are shown (Merge). N: nucleus. Yellow arrowheads indicate Ats-1 translocated to the cytoplasm of host cells. Scale bar: 5 µm. B. Percentage of HL-60 cells with P44 signal having non-colocalized (secreted) Ats-1 signal at 22, 32 or 42 h p.i. One hundred A. phagocytophilum P44-positive HL-60 cells at 22, 32 or 42 h p.i. were scored, and the percentage of cells which has Ats-1 cytoplasmic signal was determined. Data are presented as the means and standard deviations of triplicate samples. C. A 3-D shadow projection image was reconstructed based on the Z-stack data from confocal microscopy, performed for A. phagocytophilum-infected HL-60 cells at 42 h p.i. Green color: P44; Red color: Ats-1.
Figure 3.
Ats-1 localizes with mitochondria in host cells.
A and B. Triple immunofluorescence labeling of A. phagocytophilum-infected human neutrophils (A) and HL-60 cells (B) using horse anti-A. phagocytophilum (Anaplasma; Cy3, red), rabbit anti-Ats-1 (Ats-1; Alexa Fluor 488, green) and monoclonal anti-Mn-Sod (Mn-Sod; Alexa Fluor 350, gray pseudocolor). Arrowheads indicate Ats-1 and mitochondria colocalization. Scale bar: 5 µm. C and D. Negative controls for Ats-1 or Mn-Sod staining in A. phagocytophilum–infected HL-60. A. phagocytophilum–infected HL-60 cells were stained with primary antibodies, horse anti-A. phagocytophilum, and rabbit anti-Ats-1 (C), or monoclonal anti-Mn-Sod (D), then stained with secondary antibodies, Cy3-conjugated goat anti-horse IgG, Alexa Fluor 488-conjugated goat anti-rabbit IgG, and Alexa Fluor 350-conjugated goat anti-mouse IgG (gray pseudocolor). Arrowhead indicates secreted Ats-1. Scale bar: 5 µm.
Figure 4.
Mitochondrial targeting of Ats-1, and essential role of N-terminal sequence in targeting.
A-C. Double immunofluorescence labeling of A. phagocytophilum–infected HL-60 cells (A), or pAts-1-transfected RF/6A cells (B and C) using rabbit anti-Ats-1 (Ats-1; Alexa Fluor 488, green), and monoclonal anti-Mn-Sod (Mn-Sod; Alexa Fluor 555, red) (A and C), monoclonal anti-cytochrome c (Cyto c; Alexa Fluor 555, red) (B). Scale bar: 10 µm. D. Immunofluorescence labeling of RF/6A cells transfected with pAts-1ΔN17 was performed using anti–Ats-1 (Alexa Fluor 488, green). N, nucleus. Note diffuse distribution of Ats-1ΔN17 in the cytoplasm of RF/6A cell. Scale bar: 10 µm.
Figure 5.
Ats-1 is cleaved in the mitochondria.
A. Western blot analysis of the A. phagocytophilum- and mitochondria-containing pellet (Pellet) after 10,000×g centrifugation, and A. phagocytophilum purified from the pellet by Percoll density-gradient centrifugation [AP (Percoll)] using anti-Ats-1. Arrows 1 and 2 indicate full-length native Ats-1 and cleaved Ats-1, respectively. Molecular mass markers are indicated at left. B. Western blot analysis of A. phagocytophilum (Ap)-infected HL-60 cells and percoll-purified A. phagocytophilum [AP (Percoll)], using mouse monoclonal anti-P44 and rabbit anti-Mn-Sod. Molecular mass markers are indicated at left. C. Western blot analysis of pAts-1 or pAts-1ΔN17-transfected RF/6A cells using anti-Ats-1. Ats-1 was cleaved in A. phagocytophilum (Ap)-infected HL-60 cells and pAts-1-transfected cells, but not in pAts-1ΔN17-transfected cells. Arrows 1 and 2 indicate full-length Ats-1 and cleaved Ats-1, respectively. Molecular mass markers are indicated at left.
Figure 6.
Ats-1 presequence is cleaved at a specific site.
A. Schematic diagram indicating HA tag insertion and predicted cleavage sites in Ats-1 mutants. The HA tag was inserted between residues 30 and 31, 45 and 46, 60 and 61, or 72 and 73 of Ats-1. MTS, predicted mitochondrial targeting sequence, indicated by white bar. Black bar indicates cleaved Ats-1 fragments detected by Western blot analysis; Gray bar indicates the sequence between MTS and cleavage site. Dashed lines indicate undetectable (degraded) N-terminal cleaved fragment. B. Immunofluorescence labeling of RF/6A cells transfected with pAts-1 (HA45) using rabbit anti-Ats-1 [Ats-1(HA45); Alexa Fluor 488, green] and mouse monoclonal anti-Mn-Sod (Mn-Sod; Alexa Fluor 555, red). Note mitochondrial localization of Ats-1 (yellow). Scale bar: 10 µm. C. Western blot analysis using anti-Ats-1 or monoclonal anti-HA antibody was performed to examine Ats-1 cleavage in RF/6A cells transfected with recombinant plasmids encoding wild type Ats-1 or Ats-1 mutants with HA insertion at different locations. Molecular mass markers are indicated at left. D. Summarized result for Figure 6C. The molecular size for four Ats-1 mutants in Western blot analysis using anti-Ats-1 (Ats-1) or anti-HA (HA) antibody for Ats-1 (49 kDa) and cleaved Ats-1 (35, or 36 kDa). + indicates immunoreactivity; - indicates no immunoreactivity. Note the molecular mass of full-length Ats-1 or cleaved Ats-1 which has HA insertion increases 1 kDa. E. Diagram showing sequential substitution of amino acid triplets within residues 46–60 of full-length Ats-1. The wild type Ats-1 sequence at residues 46–60 was shown at the top and the mutant sequences in this region are shown below. Substituted residues were indicated in red. F. Immunofluorescence labeling of RF/6A cells transfected with pAts-1 (FYH55-57AAA) using rabbit anti-Ats-1 [Ats-1(FYH55-57AAA); Alexa Fluor 488, green] and mouse monoclonal anti–Mn-Sod (Mn-Sod; Alexa Fluor 555, red). Note mitochondrial localization of Ats-1 (yellow). Scale bar: 10 µm. G. Western blot analysis using anti–Ats-1 was performed to examine Ats-1 cleavage in RF/6A cells transfected with recombinant plasmids encoding wild type Ats-1 or the indicated Ats-1 triplet substitution mutant. Cleavage was inhibited only in cells expressing the Ats-1 (FYH55-57AAA) mutant. Molecular mass markers are indicated at left.
Figure 7.
Analysis of the import and sub-mitochondrial localization of Ats-1.
A. Ats-1, Ats-1 (55–57) and Ats-1 ΔN17 35S-labeled proteins were imported into isolated HeLa mitochondria for 45 min and treated with 50 µg/ml of protease K (PK). Mock samples contain no mitochondria. B. 35S-labeled Ats-1 was imported into isolated HeLa mitochondria for given time periods, and samples were left untreated (-PK) or treated with 50 µg/ml of protease K (+PK). Two µl of lysate of Ats-1 and Ats-1 ΔN17 was resuspended in Laemmli buffer and analyzed on the gel in addition. C. Mitochondria were left untreated (-Trypsin) or treated with 0.02 µg/µl of trypsin (+Trypsin) prior to the import. Import was then performed as in (A) and (B). Signals were quantified using ImageQuant. The import at the longest time point in -Trypsin mitochondria was set as 100%. D. HeLa cells were transfected with plasmids containing Ats-1 or Ats-1 (55–57). Mitochondria were isolated after 24 h of overexpression. Mitochondria were incubated in isotonic buffer (- SW), or were swollen in hypotonic buffer (+SW), and then were left untreated (-PK) or treated with 50 µg/ml of protease K (PK). In one sample, mitochondria were solubilized by 1% Triton X-100 (1% Triton), treated with PK and proteins were precipitated using trichloroacetic acid. In addition, 50 µg of mitochondria isolated from cells expressing Ats-1 (55–57) [HeLa Ats-1(55–57)] was resuspended in Laemmli buffer and all samples were analyzed by SDS-PAGE and Western blot, using antibodies against Mitofilin, Tim44 or Ats-1. E. Ats-1 in mitochondria behaves as a soluble protein. Ats-1 or a mutant, processing deficient form of the protein Ats-1 (55–57) were expressed in HeLa cells for 24 h. Mitochondria were isolated and subjected to carbonate extraction with 100 mM Na2CO3, pH 11.5. After centrifugation for 1 h at 100,000 g, pellet (P) and supernatant (S) fraction were separated and, together with total mitochondria, analyzed by SDS-PAGE and Western blot. Membrane was probed with Ats-1, Tom40 and Hsp60 antibodies.
Figure 8.
Ats-1 inhibits etoposide-induced apoptosis.
A-C. Triple fluorescence labeling using rabbit anti-Ats-1 [Ats-1 or Ats-1(55–57); Alexa Fluor 488, green], mouse monoclonal anti-cytochrome c (Cyto c; Alexa Fluor 555, red), and DAPI (nucleus, gray pseudocolor) in RF/6A cells transfected with pAts-1 (Ats-1) (A), pAts-1 (55–57) [Ats-1(55–57)] (B), or pGFP (GFP) (C), after treatment with etoposide. Scale bar: 10 µm. Note mitochondrial colocalization of Ats-1, a large nucleus, and a spread-out large RF/6A cell in A, and round-up shrunken RF/6A cells with condensed nuclei and presence of diffuse cytochrome c released into the cytosol in B and C. Some Ats-1 (55–57) is colocalized with cytochrome c retained in the fragmented mitochondria in B (white arrowhead). Contours of host cells are marked with white dashed lines. D. Percentage of apoptotic cells in RF/6A cells transfected with pAts-1 (Ats-1), pAts-1 (55–57) [Ats-1(55–57)], or pGFP (GFP) 1 day after etoposide treatment. Data are presented as means ± standard deviations of triplicate samples. *, significantly different compared with GFP-, or Ats-1(55–57)-expressing RF/6A cells by the Tukey's HSD test (P<0.01). E. Bax staining for RF/6A cells transfected with pAts-1 (Ats-1), pAts-1(55–57) [Ats-1(55–57)], or pGFP (GFP) 1 day after etoposide treatment. Note clumped Bax in the round-up shrunken pAts-1(55–57)- and pGFP-transfected cells, and weak diffuse Bax (almost invisible) in pAts-1-transfected cells, and absence of Bax colocalization with Ats-1 in the mitochondria. In pAts-1(55–57)-transfected cells some Bax is colocalized with Ats-1(55–57) retained in the fragmented mitochondria (white arrowhead). Bar: 10 µm. Contours of host cells are marked with white dashed lines. F. Percentage of RF/6A cells transfected with pAts-1(Ats-1), pAts-1(55–57) [Ats-1(55–57)], or pGFP (GFP), showing obvious Bax redistribution (clumping) 1 day after etoposide treatment. Data are presented as means ± standard deviations of triplicate samples. *, significantly different compared with GFP-, or Ats-1(55–57)-expressing RF/6A cells by the Tukey HSD test (P<0.01). G. Bax amount in RF/6A cells transfected with pAts-1(Ats-1), or pGFP (GFP) 1 day after etoposide treatment. Actin is used to normalize the protein loading amount. H. Western blot analysis for cleavage of PARP in RF/6A cell transfected with pAts-1 (Ats-1), pAts-1(55–57) [Ats-1(55–57)], or pGFP (GFP) after treatment with etoposide or DMSO for 12 h. Samples were subjected to probing with anti-PARP, anti-actin, or anti-Ats-1 by Western blot analysis. Full-length and cleaved PARP, actin, full-length and cleaved Ats-1 are indicated with arrows. Relative density ratios of cleaved PARP/actin bands, or total Ats-1 (full length, cleaved and degraded Ats-1)/actin bands are shown below each lane in etoposide-treated group.
Figure 9.
Ats-1 inhibits Bax-induced apoptosis in yeast.
A. Mitochondrial colocalization of Ats-1 and Ats-1(55–57) in S. cerevisiae. pYAts-1 (Ats-1), pYAts-1(55–57) [Ats-1(55–57)], or pGADT7 AD(GADT7)–transformed YPH499 yeast cells were loaded with MitoTracker Red, and subjected to immunostaining using rabbit anti-Ats-1, and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Scale bar: 5 µm. B. Expression of Ats-1, or Ats-1(55–57) in S. cerevisiae. The cell lysates of YPH499 cells transformed with pYAts-1 (Ats-1), pYAts-1(55–57) [Ats-1(55–57)], or pGADT7 AD(GADT7) were subjected to Western blot analysis using anti-Ats-1. Note 35-kDa mature Ats-1 from wild type Ats-1 and abnormal cleavage of Ats-1(55–57). C. Ats-1 partially rescues S. cerevisiae from Bax-induced growth arrest. Yeast YPH499 cells were co-transformed with pBax, and pGADT7 AD (GADT7), pYAts-1(55–57) [Ats-1(55–57)], or pYAts-1(Ats-1). Recombinant yeast cells were cultured in SG medium containing galactose to induce Bax expression. The number of viable yeast cells was determined by plate count technique. The numbers of viable cells at day 5 after Bax induction were compared to the viable cells at day 0. Data are presented as means and standard deviations of three independent experiments. * and **, Significantly different among each other by Tukey's HSD test (P<0.05). D. Translocation of Bax to mitochondria in S. cerevisiae. After induction to express Bax for 12 h, the total cell lysates and mitochondria isolated from YPH499 cells co-transformed with pBax, and pGADT7 AD (GADT7), pYAts-1(55–57) [Ats-1(55–57)], or pYAts-1(Ats-1), were subjected to Western blot analysis using anti-Bax, and anti- S. cerevisiae Porin. Relative density ratios of Bax/Porin bands are shown below each lane.