Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability - PubMed (original) (raw)
Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability
Jing Zou et al. Biochem J. 2013.
Abstract
The mitochondrion-associated protein LRPPRC (leucine-rich pentatricopeptide repeat-containing) interacts with one of the microtubule-associated protein family members MAP1S (microtubule-associated protein 1 small form), originally named C19ORF5 (chromosome 19 open reading frame 5), to form a complex. MAP1S interacts with LC3 (light chain 3), the mammalian homologue of yeast autophagy marker ATG8 and one of the most important autophagy markers in mammalian cells, and helps the attachment of autophagosomes with microtubules for trafficking and recruitment of substrate mitochondria into autophagosomes for degradation. MAP1S activates autophagosomal biogenesis and degradation to remove misfolded/aggregated proteins and dysfunctional organelles such as mitochondria and suppress oxidative stress-induced genomic instability and tumorigenesis. Previously, various studies have attributed LRPPRC nucleic acid-associated functions. Instead, in the present study, we show that LRPPRC associates with mitochondria, interacts with Beclin 1 and Bcl-2 and forms a ternary complex to maintain the stability of Bcl-2. Suppression of LRPPRC leads to reduction in mitochondrial potential and reduction in Bcl-2. Lower levels of Bcl-2 lead to release of more Beclin 1 to form the Beclin 1-PI3KCIII (class III phosphoinositide 3-kinase) complex to activate autophagy and accelerate the turnover of dysfunctional mitochondria through the PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) pathway. The activation of autophagy induced by LRPPRC suppression occurs upstream of the ATG5-ATG12 conjugate-mediated conversion of LC3-I into LC3-II and has been confirmed in multiple mammalian cell lines with multiple autophagy markers including the size of GFP-LC3 punctate foci, the intensity of LC3-II and p62 protein and the size of the vacuolar structure. The activated autophagy enhances the removal of mitochondria through lysosomes. LRPPRC therefore acts to suppress the initiation of basal levels of autophagy to clean up dysfunctional mitochondria and other cellular debris during the normal cell cycle.
Figures
Figure 1. Depletion of LRPPRC leads to a decrease in mitochondrial potential
(A) Immunostaining analysis showing the co-localization of antibody-stained LRPPRC (green) with MitoTracker-labelled mitochondria (red) in HeLa cells. (B) Plot of intensities of LRPPRC (green) and mitochondrial signals (red) presented in (A). (C) Analysis of the impact of LRPPRC depletion on the mitochondrial potentials labelled with MitoTracker (red) in HeLa cells treated with mock or LRPPRC siRNA for 72 h. LRPPRC were labelled with anti-LRPPRC antibody (green). (D) Comparison of MitoTracker intensities between two neighbouring cells with (right) and without (left) LRPPRC suppressed. (E) Comparison of cytochrome c signals (red) between cells retaining normal levels of LRPPRC (*, green) and cells with LRPPRC completely silenced (∧). (F) Immunoblot (IB) of lysates from HeLa cells treated without siRNA (Mock) or with siRNA specifically targeting LRPPRC (LRPPRC). Molecular masses are indicated in kDa. Scale bars in (A) and (C)–(E), 10 μm.
Figure 2. Suppression of LRPPRC leads to enhancement of basal levels of autophagy in HeLa cells stably expressing GFP–LC3
(A) Immunostaining of HeLa cells stably expressing GFP–LC3 treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) for 72 h in the absence (Ctrl) or presence of lysosomal inhibitor NH4Cl (20 mM overnight before harvest). (B) Enlarged views of GFP–LC3 punctate foci under similar treatments as shown in (A). (C) Quantification of GFP–LC3-labelled autophagosomes. The total area occupied by GFP–LC3 punctate foci is the mean±S.D. for ten randomly selected images in a field of 512 pixels×512 pixels. The significance of differences was determined by Student's t test. (D) Immunoblot (IB) of lysates from HeLa cells stably expressing GFP–LC3 similarly treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence of lysosomal inhibitor NH4Cl. Molecular masses are indicated in kDa.
Figure 3. Suppression of LRPPRC leads to enhancement of basal levels of autophagy in native HeLa cells
(A) Immunoblot analyses of LC3 isoforms in lysates from HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence of lysosomal inhibitor NH4Cl. Molecular masses are indicated in kDa. (B) Plots of relative intensities of LC3-I and LC3-II bands. The LC3-I and LC3-II intensities in samples treated with mock siRNA were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. *_P_≤0.05. (C) Transmission electron microscopy imaging of HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of lysosomal inhibitor bafilomycin A1. *, autophagy vacuoles. (D) Plot of percentages of area occupied by autophagy vacuoles in the transmission electron microscopy images. Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. *_P_≤0.05. (E) Immunoblot (IB) analyses of p62 levels in lysates from HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of bafilomycin A1. (F) Plots of relative intensities of p62. The p62 intensities in samples treated with mock siRNA were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. *_P_≤0.05. (G) Immunostaining analysis of p62 levels in HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of bafilomycin A1.
Figure 4. Overexpression of LRPPRC leads to inhibition of basal levels of autophagy in COS7 cells
(A) Immunoblot analysis of LRPPRC protein levels in different types of cells. Lysates with the same amount of total proteins prepared from HeLa, HEK-293T and COS7 cells were compared. Molecular masses are indicated in kDa. (B) Immunoblot analyses of LC3 levels in lysates from COS7 cells overexpressing GFP or GFP–LRPPRC in the absence (−) or presence (+) of lysosomal inhibitor bafilomycin A1 (BAF). Expression levels of LRPPRC were confirmed by immunoblotting with antibodies against LRPPRC (top panel) or GFP (second panel). Molecular masses are indicated in kDa. (C) Plots of relative intensities of LC3-I and LC3-II bands with representative images shown in (B). The intensities in cells expressing GFP in the absence of lysosomal inhibitor were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. *_P_≤0.05.
Figure 5. Suppression of LRPPRC leads to activation of mitophagy in HeLa cells
(A) Fluorescence imaging analysis of the co-localization of GFP–LC3 with the mitochondrial marker Tom20. HeLa cells stably expressing GFP–LC3 treated with random siRNA (Mock) or LRPPRC siRNA (LRPPRC) for 72 h and with (BAF) or without (Ctrl) bafilomycin A1 in the last 12 h. (B) Plots of ratio (percentages) of GFP–LC3-associated Tom20 to total Tom20 (upper panel) or Tom20-associated GFP–LC3 to total GFP–LC3 (lower panel) with representative images shown in (A). Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. *_P_≤0.05. (C) Immunostaining analysis of Tom20 levels in HeLa cells treated with random siRNA (Mock) or LRPPRC-specific siRNA (LRPPRC) in the absence (Ctrl) or presence (BAF) of bafilomycin A1. (D) Immunoblot (IB) analyses of Tom20 levels in lysates from HeLa cells treated with LRPPRC siRNA for 72 h and bafilomycin A1 (BAF) in the last 12 h. (E) Plots of relative intensities of Tom20 bands with representative images shown in (D). Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. *_P_≤0.05. (F) Fluorescence imaging analysis showing the co-localization of Tom20 with lysosomal marker LAMP1. HeLa cells treated with random siRNA (Mock) or LRPPRC siRNA (LRPPRC) for 72 h and with (BAF) or without (Ctrl) bafilomycin A1 in the last 12 h. Scale bars, 10 μm. (G) Plots of ratio (percentages) of LAMP1-associated Tom20 to total Tom20 with representative images shown in (F). Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. *_P_≤0.05. (H) Fluorescence imaging analysis showing the co-localization of Tom20 with lysosomal marker LAMP2. HeLa cells treated with random siRNA (Mock) or LRPPRC siRNA (LRPPRC) for 72 h and with (BAF) or without (Ctrl) bafilomycin A1 in the last 12 h. (I) Plots of ratio (percentages) of LAMP2-associated Tom20 to total Tom20 with representative images shown in (H). Results are means±S.D. of at least three repeats and the differences were compared using Student's t test. *_P_≤0.05.
Figure 6. Activation of autophagy resulting from LRPPRC depletion occurs upstream of the conversion of LC3-I into LC3-II
(A) Immunoblot analyses showing the impact of LRPPRC on the levels of p27 and ATG5. HeLa cells were treated with siRNA specific to LRPPRC or p27 either individually or in combination for 72 h in the absence (Ctrl) or presence (BAF) of bafilomycin A1 (10 nM overnight overnight before harvest). (B) Immunoblot analyses showing the impact of p27 and ATG5 on LRPPRC-suppressed autophagy initiation. HeLa cells were treated with siRNA specific to p27, ATG5 and/or LRPPRC individually or in combination for 72 h in the absence (Ctrl) or presence (BAF) of bafilomycin A1 (10 nM overnight overnight before harvest). Molecular masses are indicated in kDa.
Figure 7. LRPPRC regulates the levels of Bcl-2
(A) Immunoblot (IB) analyses of lysates prepared from HeLa or HEK-293T cells treated with mock or LRPPRC siRNA for 72 h showing the impact of LRPPRC depletion on levels of proteins controlling autophagy initiation through the PI3K/Akt/mTOR pathway. Molecular masses are indicated in kDa. (B) Plots of relative intensities of Beclin 1, PI3KCIII and Bcl-2 bands as shown in (A). The intensities in samples treated with mock siRNA were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. *_P_≤0.05. (C) Immunoblot (IB) analyses of lysates prepared from COS7 cells overexpressing LRPPRC showing the impact of LRPPRC overexpression on levels of proteins controlling autophagy initiation through the PI3K/Akt/mTOR pathway. Expression levels of LRPPRC were confirmed by immunoblot with antibodies against LRPPRC (top panel) or GFP (second panel). Molecular masses are indicated in kDa. (D) Plots of relative intensities of Beclin 1, PI3KCIII and Bcl-2 bands in COS7 cells overexpressing LRPPRC as shown in (C). The intensities in cells overexpressing GFP were set to 1. Results are means±S.D. of at least three repeats and the differences were compared using a paired Student's t test. *_P_≤0.05.
Figure 8. LRPPRC interacts with Beclin 1 and Bcl-2 and prevents Beclin 1 from forming an autophagy-inducing complex with PI3KCIII
(A) Representative result of co-immunoprecipitation analyses of the LRPPRC–Beclin 1 interaction. The same amount of HeLa cell lysates was used to perform immunoprecipitation with the same amount of anti-LRPPRC antibody or mouse IgG control. (B) Representative result of co-immunoprecipitation analyses of the LRPPRC–Bcl-2 interaction. The same amount of HeLa cell lysates was used to perform immunoprecipitation with the same amount of anti-LRPPRC antibody or mouse IgG control. (C) Representative result of co-immunoprecipitation analyses of the interaction of Bcl-2 with LRPPRC and Beclin 1. The same amount of HeLa cell lysates was used to perform immunoprecipitation with the same amount of anti-Bcl-2 antibody or mouse IgG control. (D) Representative result of co-immunoprecipitation analyses of interaction of LRPPRC with PI3KCIII. The same amount of HeLa cell lysates were used to perform immunoprecipitation with the same amount of anti-LRPPRC antibody or mouse IgG control. (E and F) Co-immunoprecipitation analyses of the impact of LRPPRC depletion on Beclin 1–Bcl-2 and Beclin 1–PI3KCIII interactions. Lysates containing equal amounts of total proteins prepared from HeLa (E) or HEK-293T cells (F) treated with mock or LRPPRC siRNA were immunoprecipitated with anti-Beclin 1 or control IgG antibody and the precipitates were immunoblotted with antibodies against Beclin 1, Bcl-2 and PI3KCIII. (A)–(F) IB, immunoblot; IP, immunoprecipitation. Molecular masses are indicated in kDa. (G) Impact of LRPPRC depletion on the interaction of Beclin 1 with Bcl-2 or PI3KCIII.
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