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. 2019 May 1;30(10):1138-1146.
doi: 10.1091/mbc.E19-01-0044. Epub 2019 Mar 6.

Retromer Facilitates the Localization of Bcl-xL to the Mitochondrial Outer Membrane

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Free PMC article

Retromer Facilitates the Localization of Bcl-xL to the Mitochondrial Outer Membrane

Trey Farmer et al. Mol Biol Cell. .
Free PMC article

Abstract

The anti-apoptotic Bcl-2 family protein Bcl-xL plays a critical role in cell survival by protecting the integrity of the mitochondrial outer membrane (MOM). The mechanism through which Bcl-xL is recruited to the MOM has not been fully discerned. The retromer is a conserved endosomal scaffold complex involved in membrane trafficking. Here we identify VPS35 and VPS26, two core components of the retromer, as novel regulators of Bcl-xL. We observed interactions and colocalization between Bcl-xL, VPS35, VPS26, and MICAL-L1, a protein involved in recycling endosome biogenesis that also interacts with the retromer. We also found that upon VPS35 depletion, levels of nonmitochondrial Bcl-xL were increased. In addition, retromer-depleted cells displayed more rapid Bax activation and apoptosis. These results suggest that the retromer regulates apoptosis by facilitating Bcl-xL's transport to the MOM. Importantly, our studies suggest a previously uncharacterized relationship between the machineries of cell death/survival and endosomal trafficking.

Figures

FIGURE 1:
FIGURE 1:
Bcl-xL resides in a protein complex with members of the retromer and DRP1. (A) HeLa cell lysates were subjected to immunoprecipitations with anti-VPS26, anti–Bcl-xL, or control IgG, and immunoblotted with antibodies against Drp1, VPS26, and Bcl-xL. Three different exposures of the same immunoblot are depicted: low, medium, or high exposure. Gels depicted are representative of three independent experiments showing similar results. Densitometric analysis from these experiments shows that 1) compared with the level of VPS26 precipitated with anti-VPS26 (defined as 100%), 41–66% of VPS26 precipitates with anti–Bcl-xL, and 2) the level of DRP1 precipitated by anti–Bcl-xL ranges from ∼50 to 90% of that precipitated by anti-VPS26. (B) HeLa cell lysates were subjected to immunoprecipitations with anti–Bcl-xL, anti–MICAL-L1, or control IgG, and immunoblotted with antibodies against MICAL-L1, VPS35, VPS26, and Bcl-xL. Gel depicted is representative of three individual experiments showing similar results. Densitometric analysis from these experiments shows that 1) the ratio of VPS26:Bcl-xL precipitated with anti–Bcl-xL is 0.660+/− 0.110, which is very similar to the ratio of VPS35:Bcl-xL precipitated with anti–Bcl-xL (0.6866+/− 0.169), and 2) the ratio of VPS26:MICAL-L1 precipitated with anti–MICAL-L1 (0.589+/− 0.215) is similar to the ratio of VPS35:MICAL-L1 precipitated with anti–MICAL-L1 (0.607+/− 0.129). (C) Efficacy of DRP1 depletion is demonstrated by immunoblotting lysates from Mock- or DRP1-depleted HeLa cells with anti-DRP1, and using GAPDH as a loading control. (D) Densitometric quantification of DRP1 protein levels in either Mock- or DRP1-siRNA treatment. Error bars denote SD. p values were determined by the Student’s one-tailed t test. n = 3. (E) HeLa cells were treated with either Mock- or DRP1-siRNA, immunoprecipitated with antibodies against Bcl-xL, and immunoblotted with antibodies against VPS35, VPS26, and Bcl-xL. Gel depicted is representative of three individual experiments showing similar results. (F) Densitometric quantification of VPS35 or VPS26 protein levels immunoprecipitated by anti–Bcl-xL in the presence or absence of DRP1. Error bars denote SD. p values were determined by the Student’s one-tailed t test. n = 3.
FIGURE 2:
FIGURE 2:
Bcl-xL localizes to endocytic vesicles containing the retromer. (A) RPE1 cells were transfected with the N-terminal 10 residues of the mitochondrial outer membrane protein (Tom20) tagged with mCherry (mCh-Tom20; red), and immunostained with VPS26 (blue) and Bcl-xL (green). Images shown are 3D snapshots of serial z-sections. Channels were split, showing the individual protein localization patterns (right panels). Regions of interest are highlighted with dashed boxes labeled as 1 or 2, and they correspond to the inset images depicted in B and C. Scale bar = 10 μm. (B) Inset area 1 from A. Arrows denote vesicles containing VPS26 (blue) and Bcl-xL (green) but lack mCh-Tom20 (red). Channels were split, showing the individual protein localizations. Scale bar = 2 μm. (C) Inset area 2 from A. Dashed circles show areas where Bcl-xL (green) is colocalized with mCh-Tom20 (red), but not VPS26 (blue). Channels were split, showing the individual protein localizations. Scale bar = 2 μm. Images portrayed are representative of three independent experiments (quantified in D). (D) The colocalization threshold analysis tool in Fiji ImageJ was used to quantify the colocalization between Bcl-xL and mCh-Tom20, Bcl-xL and VPS26, or mCh-Tom20 and VPS26. Data are presented as a mean, and error bars indicate SD. n = 3. (E) A single representative RPE1 cell transfected with mCh-Tom20 (red), and immunostained with VPS26 (blue) and Bcl-xL (green). The image shown is a 3D snapshot of serial z-sections. Blue arrows depict VPS26 and Bcl-xL colocalization, whereas yellow arrows depict Bcl-xL and Tom20 colocalization. Scale bar = 10 μm. (F–H) Individual two-channel images from E are shown depicting the colocalization between Tom20 and Bcl-xL (F), VPS26 and Tom20 (G), and between Bcl-xL and VPS26 (H).
FIGURE 3:
FIGURE 3:
Loss of VPS35 or MICAL-L1 leads to increased nonmitochondrial Bcl-xL. (A) RPE1 cells were subjected to Mock-, VPS35-, or MICAL-siRNA, immunostained with antibodies against Tom20 (green) and Bcl-xL (red), and serial z-sections were obtained. The images depicted are 3D snapshots. Dashed regions of interest correspond to the insets in the three right-hand panels. Inset channels were split, showing the individual protein localization patterns. Scale bar = 10 μm, (2 μm; inset). (B) Quantification of the mean number of nonmitochondrial-associated Bcl-xL structures upon Mock-, VPS35-, and MICAL-L1-siRNA treatment. Error bars denote SD. p values were determined by the Student’s one-tailed t test. n = 3. (C) Efficacy of the VPS35-depletion is demonstrated by immunoblotting lysates from Mock- or VPS35-depleted RPE1 cells, with GAPDH as a loading control. (D) Efficacy of MICAL-L1-depletion is demonstrated by immunoblotting lysates from Mock- or MICAL-L1–depleted RPE1 cells using GAPDH as a loading control. (E) HeLa cells were treated with either Mock- or VPS35-siRNA, homogenized, and subject to immunofractionation with anti-Tom20 to generate an enriched mitochondrial fraction (Mt) and a nonmitochondrial fraction (Non-Mt). The fractions were separated by SDS–PAGE and immunoblotted with anti–Bcl-xL and anti-Tom20. (F) Densitometric quantification of the ratio of nonmitochondrial Bcl-xL vs. mitochondrial Bcl-xL in either Mock- or VPS35- siRNA treatment. Error bars denote SD. p values were determined by the Student’s one-tailed t test. n = 3.
FIGURE 4:
FIGURE 4:
The rate of Bax activation at the mitochondrial membrane is enhanced in cells lacking VPS35. (A) CRISPR/Cas9 HCT 116 cells lacking endogenous Bak and Bax, but expressing stably transfected GFP-Bax, were subject to Mock- or VPS35-siRNA knockdown, with or without staurosporine (STS) treatment for 60 min. Cells were fixed and immunostained with anti-Tom20 (red). For micrographs representing the STS treatment, only GFP-Bax is shown. Scale bar = 10 μm. (B) Quantification of the mean percentage of Mock- or VPS35-siRNA–treated cells displaying GFP-Bax activation upon STS treatment. Error bars represent SD. p value was determined by the Student’s one-tailed t test. n = 3. (C) Efficacy of the VPS35-siRNA treatment is demonstrated by immunoblotting lysates from Mock- or VPS35-depleted CRISPR/Cas9 HCT 116 cells with anti-VPS35. GAPDH was used as a loading control. (D) CRISPR/Cas9 HCT 116 cells lacking endogenous Bak and Bax, but expressing stably transfected GFP-Bax, were subject to either Mock- or VPS35-siRNA treatment for 48 h, and treated acutely with STS for 0, 30, or 60 min. Lysates from each treatment were analyzed by immunoblotting for Parp1 to assess cleavage over time, and immunoblotting with anti-VPS35 was used to verify the siRNA treatment efficacy. GAPDH was used as a loading control. (E) Densitometric representation of the data from D was done using ImageJ to calculate the ratio of Parp1:GAPDH between Mock- and VPS35-siRNA–treated cells. Data are presented as a mean, and error bars indicate SD. p values were determined by the Student’s one-tailed t test. n = 3.
FIGURE 5:
FIGURE 5:
Model for the role of retromer in regulating Bcl-xL’s translocation to the mitochondrial membrane and impact on staurosporine-induced apoptosis. Under physiological conditions (top), staurosporine treatment induces Bax translocation to the mitochondrial membrane. Because Bcl-xL is constitutively transported to the MOM, Bax pore formation is inhibited and slowed by Bcl-xL, but when sufficient Bax pore formation occurs, Cyt c is released and apoptosis occurs. Upon VPS35 knockdown (bottom), there is impaired retromer complex generation and decreased constitutive transport of Bcl-xL to the MOM. Accordingly, upon staurosporine treatment there is less inhibition of Bax by Bcl-xL, leading to more rapid Bax pore formation and an increased rate of apoptosis.

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