Fig 1.
mtFAS generates octanoyl-ACP, that enters the lipoic acid biosynthesis pathway. The octanoyl moiety is then transferred from ACP to H or E2 proteins. Subsequently, insertion of two sulfur atoms occurs on the octanoyl moiety to generate lipoylated H or E2 proteins. 2-KGDH, α-ketoglutarate dehydrogenase; 2-OADH, 2-oxoadipate dehydrogenase; ACP, acyl carrier protein; BCKDH, branched-chain ketoacid dehydrogenase; GCS, glycine cleavage system; LA, lipoic acid; LIAS, lipoic acid synthetase; LIPT1, lipoyl(octanoyl) transferase 1; LIPT2, lipoyl(octanoyl) transferase 2; mtFAS, mitochondrial fatty acidynthesis; PDH, pyruvate dehydrogenase.
Fig 2.
LIPT2 and LIPT2-EYFP target the mitochondrion.
(A) Immunodetection of endogenous LIPT2 (~25 kDa) in mitochondrial, cytosolic and microsomal fractions obtained from HEK 293 Phoenix cellular proteins. Alpha-tubulin (55 kDa) and ox-Phos-complex IV (35–40 kDa) were used as markers of the non-mitochondrial (cytosolic and microsomal) and mitochondrial fractions, respectively. The image is representative of three independent experiments. (B) From left to right: fluorescent signal of EYFP (green) and the mitochondrial marker (magenta) in HeLa cells expressing EYFP, EYFP-LIPT2 or LIPT2-EYFP for 72 hours, corresponding merge image and scatter plot. Scale bar: 20 μm. Pearson’s correlation coefficient, overlap coefficient and co-localization rate (%) referred to the co-localization of EYFP, EYFP-LIPT2 or LIPT2-EYFP and the mitochondrion determined in HeLa cells (C), 24, (D), 48 and (E), 72 hours after transfection. (n) indicates the number of cells. **: p<0.01, ***: p<0.001, one-way ANOVA with Bonferroni’s post-test.
Fig 3.
Removal of amino acids 1–31 prevents the targeting of LIPT2 to the mitochondrion.
(A) From left to right: fluorescent signal of EYFP (green) and the mitochondrial marker (magenta) in HeLa cells expressing LIPT2-EYFP void of the amino acids 1–31 (ΔmitotagLIPT2-EYFP) for 72 hours, corresponding merge image and scatter plot. Scale bar: 20 μm. (B) Pearson’s correlation coefficient, overlap coefficient and co-localization rate (%) referred to the co-localization of ΔmitotagLIPT2-EYFP and the mitochondrion determined in HeLa cells 72 hours after transfection. (n) indicates the number of cells. ***: p<0.001, one-way ANOVA with Bonferroni’s post-test.
Fig 4.
Amino acids 1–31 of LIPT2 target EYFP to the mitochondrion.
(A) Fluorescent signal of EYFP, mitotag-EYFP, mitotag-ΔATG-EYFP after 24 (left), 48 (middle) and 72 hours (right) transfection in HeLa cells. Scale bar: 20 μm. (B) Pearson’s correlation coefficient, overlap coefficient and co-localization rate (%) referred to the co-localization of EYFP, mitotag-EYFP, mitotag-ΔATG-EYFP and the mitochondrion determined in HeLa cells 72 hours after transfection. (n) indicates the number of cells. **: p<0.01, ***: p<0.001, one-way ANOVA with Bonferroni’s post-test.
Fig 5.
Apoptotic volume decrease (AVD) current is stimulated in EYFP-LIPT2 expressing cells.
(A) Original current recordings obtained in whole-cell configuration from HEK 293 Phoenix cells kept in isotonic bath solution and stimulated with the pulse protocol represented in the inset. Cells were transfected for 48 hours with a fluorescent transfection marker without (control) or with LIPT2 or the fusion proteins LIPT2-EYFP or EYFP-LIPT2, as indicated. (B) Current density (pA/pF) to voltage (mV) relationship of control, LIPT2, LIPT2-EYFP and EYFP-LIPT2 expressing cells. (n) indicates the number of cells. **: p<0.01 compared to control, LIPT2 and LIPT2-EYFP, unpaired Student’s t test.
Fig 6.
IClswell current is over-stimulated in EYFP-LIPT2 and ΔmitotagLIPT2 expressing cells.
(A) Original current recordings obtained in whole-cell configuration from HEK 293 Phoenix cells in hypertonic (hyper) and hypotonic (hypo) bath solution following stimulation with the pulse protocol represented in the inset. Cells were transfected for 48 hours with EYFP-LIPT2 or LIPT2-EYFP, as indicated. (B) Current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in cells expressing EYFP-LIPT2 or LIPT2-EYFP. C, current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in cells expressing LIPT2 or ΔmitotagLIPT2. In B, left, ***: p<0.001 at all applied voltages except for 0 mV compared to LIPT2-EYFP, hypo, unpaired Student’s t test, and right, p<0.001 when time >160 sec, unpaired Student’s t test. In C, left, *: p<0.05 at all applied voltages except for 0 mV compared to LIPT2, hypo, unpaired Student’s t test, and right, p<0.05 when time >360 sec and <660 sec, unpaired Student’s t test. (n) indicates the number of cells.
Fig 7.
IClswell current is not affected following alteration of LIPT2 expression.
(A) Current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in HEK 293 Phoenix cells transfected for 48 hours with LIPT2 or a transfection marker (control). (B) Levels of the transcript of LIPT2 detected by RT-PCR in HEK 293 Phoenix cells transfected for 48 hours with siRNA #1, 2 and 3 or control siRNA (control) and normalized for the levels of the transcript of the housekeeping gene β-ACTIN. Gene silencing of 7, 48 and 25% was obtained, respectively. (C) Current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in cells transfected with siRNA #2 or a control siRNA (control). n.s., not significant, **, ***: p<0.01, p<0.001, unpaired Student’s t test. (n) in A and C indicates the number of cells. In B, n = 3 for each condition.
Fig 8.
Apoptosis is induced in EYFP-LIPT2 and ΔmitotagLIPT2 expressing cells.
(A) Representative immunodetection of calreticulin in whole cell lysates from HEK 293 Phoenix cells 48 hours after transfection with the indicated constructs or from cells treated with the vehicle or 20 μM staurosporine for 4 hours. The lower panels correspond to the signal of the housekeeping protein GAPDH. (B) The expression levels of calreticulin were quantified by densitometry and normalized to GAPDH. n = 3, *: p<0.05, **: p<0.01, ***: p<0.001, two-tailed, unpaired Student’s t test. (C) Immunodetection of cleaved caspase-3 and the housekeeping protein β-actin in whole cell lysates from HEK 293 Phoenix cells 48 hours after transfection with the indicated constructs or from cells treated with the vehicle or 20 μM staurosporine for 4 hours. The image is representative of three independent experiments. (D) From left to right: fluorescent signal of DAPI (cyan), TUNEL (magenta) and corresponding merge image of HEK 293 Phoenix cells expressing EYFP-LIPT2 (top), LIPT2-EYFP (middle), and ΔmitotagLIPT2 (bottom) for 48 hours. Scale bar: 50 μm. (E) Normalized TUNEL signal intensity of cells transfected with the indicated constructs or treated overnight with the vehicle or 2 μM staurosporine. 12≤n≤25 indicates the number of imaging fields from 3 independent experiments. *: p<0.05, one-way ANOVA with Bonferroni’s post-test.
Fig 9.
Mitochondrial membrane potential depolarization is observed in EYFP-LIPT2 and ΔmitotagLIPT2 expressing cells.
(A) From left to right: fluorescence intensity of Hoechst (nucleus), Mito Tracker Deep Red (mitochondrial potential) and corresponding merge image (nucleus in cyan and mitochondria in magenta) of HEK 293 Phoenix cells treated with the vehicle (top panels) or the uncoupling agent FCCP (bottom panels) for 30 minutes. Scale bar: 10 μm. (B) Mitochondrial membrane potential in vehicle and FCCP-treated cells. (n) refers to the number of fields imaged. ***: p<0.001, two-tailed, unpaired, Student’s t test. (C) Fluorescence intensity of Mito Tracker Deep Red (mitochondrial potential) in cells expressing the indicated constructs for 48 hours and the corresponding merge image, with transfected cells (green) within a ROI, mitochondria in magenta and nuclei counterstained with Hoechst (cyan). Scale bar: 10 μm. (D-F) Mitochondrial membrane potential determined 24, 48 and 72 hours after transfection, respectively. (n) indicates the number of cells. *: p<0.05, **: p<0.01, ***: p<0.001, one-way ANOVA with Bonferroni’s post-test.