Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Feb 18;33(4):282-95.
doi: 10.1002/embj.201385902. Epub 2014 Jan 20.

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

Gian-Luca McLelland  1 , Vincent Soubannier, Carol X Chen, Heidi M McBride, Edward A Fon
Affiliations

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

Gian-Luca McLelland et al. EMBO J. .

Abstract

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Parkin promotes the biogenesis of cargo-selective MDVs in response to mitochondrial ROS

  1. Schematic depicting the location of relevant protein markers relative to the mitochondrion, as well as the site of action of pharmacological agents used.

  2. HeLa cells expressing GFP-parkin (green), OCT-DsRed2 (red) and CFP-Drp1K38E were left untreated (untreated) or treated for 2 h with 10 μM CCCP (CCCP), then fixed and stained for TOM20 (blue). Scale bars, 30 μm.

  3. Cells prepared as in (B) were treated with 50 μM antimycin A for 2 h. Arrows indicate OCT-DsRed2-positive/TOM20-negative MDVs that colocalize with GFP-parkin, while circles indicate MDVs that are parkin-negative. Arrowheads indicate “nascent” vesicles, adjacent to mitochondria, exhibiting cargo selectivity and parkin recruitment. Open arrowheads indicate parkin-negative MDVs containing the reciprocal cargo (TOM20-positive/OCT-DsRed2-negative). Cell boundaries are delineated in the GFP-parkin single-channel image. Scale bars, 30 μm.

Figure 2
Figure 2
Wild-type parkin, but not PD-associated parkin mutants, promotes MDV biogenesis

  1. Quantification of the number of OCT-DsRed2-positive/TOM20-negative vesicles in cells expressing GFP, GFP-parkinWT, GFP-parkinR42P, GFP-parkinK211N, or GFP-parkinC431F, treated with or without antimycin A (anti A) as in (C); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 49–68 cells in 2–3 experiments); ns, not significant; **P < 0.01; ***P < 0.001).

  2. Structure of parkin, with PD-linked residues from (D) highlighted in dark gray (PDB ID: 4K95). Ubl, ubiquitin-like domain (red); RING0, RING0 domain (green); RING1, RING1 domain (cyan); IBR, in-between RING (magenta); REP, repressor element of parkin (yellow); RING2, RING2 domain (salmon).

  3. HeLa cells expressing various GFP-parkin mutant contructs (green), pOCT-DsRed2 (red) and CFP-Drp1K38E were treated with 50 μM antimycin A for 2 h, then fixed and immunostained against TOM20. Arrows indicate matrix-positive/TOM20-negative structures colocalizing with GFP-parkin. Scale bars, 30 μm.

  4. Immunoblot of whole-cell lysates of HeLa cells expressing various GFP-parkin constructs, treated with 50 μM antimycin A (anti A, +) or DMSO (−) for 2 h.

Source data are available online for this figure.
Figure 3
Figure 3
Parkin-dependent MDVs incorporate endogenous mitochondrial cargo

  1. Representative immunoblot of whole-cell lysates from U2OS:GFP and U2OS:GFP-parkin cells transfected with non-targeting siRNA or siRNA targeting Drp1 (siDrp1).

  2. U2OS:GFP-parkin cells transfected with siRNA targeting Drp1 were treated with DMSO (upper panels) or 25 μM antimycin A (anti A, lower panels) for 90 min, then fixed and immunostained against PDH (red) and TOM20 (blue). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm (first panels on left) and 2 μm.

  3. Quantification of PDH-positive/TOM20-negative vesicles in U2OS:GFP and U2OS:GFP-parkin cells transfected with the indicated siRNA, treated with DMSO or antimycin A (anti A) as in (B); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 48–85 cells in 2–3 experiments); ns, not significant; ***P < 0.001).

  4. 5 μm-long profile of the parkin-positive vesicle and adjacent mitochondrial tubule depicted in the antimycin A-treated cell from (B) (left). Increasing position values on the x-axis of the fluorescence intensity plot (right) correspond to moving from the top to the bottom of the profile. *MDV indicated by an arrow in (B).

  5. Quantification of the distance between the centres of GFP-parkin-negative (black bar) or -positive (gray bar) vesicles and the edge of the nearest mitochondrial tubule for the vesicles quantified in antimycin A-treated U2OS:GFP-parkin cells transfected with siDrp1 in (C). Error bars represent the mean ± s.e.m.; *P < 0.05 (obtained by Student's t-test).

  6. U2OS:GFP-parkin cells transfected with siRNA targeting Drp1 (siDrp1) were treated with 25 μM antimycin A for 90 min prior to fixation. Samples were immunostained against TOM20 (blue) and the indicated mitochondrial marker (red). TOM20-negative MDVs containing the specified cargo and colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Arrowheads show lack of colocalization between GFP-parkin and the indicated mitochondrial marker. Scale bars, 20 μm (first two columns of panels) and 2 μm (remaining columns of panels).

  7. Quantification of TOM20-negative structures that stained positively for the indicated cargo in U2OS:GFP (GFP) and U2OS:GFP-parkin (GFP-parkin) cells, transfected with siRNA targeting Drp1, treated with antimycin A (anti A); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 24–59 cells in two experiments; ns, not significant; ***P < 0.001).

Source data are available online for this figure.
Figure 4
Figure 4
Parkin-dependent MDVs are targeted to lysosomes independently of autophagy

  1. HeLa cells, transfected with siRNA targeting Drp1 and GFP-parkin (green), were treated with 25 μM antimycin A for 90 min following a 30-min pretreatment with 50 nM bafilomycin A1, then fixed and immunostained against PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bar, 30 μm.

  2. Quantification of PDH E2/E3bp-positive/TOM20-negative vesicles in HeLa cells treated with antimycin A in the presence of the lysosomal inhibitors bafilomycin A1 or pepstatin A and E-64d; both total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Bars represent the mean ± s.e.m. P-values are given for total vesicle number (n = 19 to 120 cells in 2–3 experiments; **P < 0.01; ***P < 0.001).

  3. HeLa cells, transfected with GFP-parkin (green) and siRNA targeting Drp1 and Atg5, were treated with 25 μM antimycin A for 90 min, then fixed, immunostained for PDH E2/E3 bp (red) and TOM20 (blue), and counterstained for Hoescht (gray). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Scale bars, 20 μm.

  4. Quantification of PDH E2/E3 bp-positive/TOM20-negative vesicles from HeLa cells transfected with the indicated siRNA; both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P-values are given for the total vesicle number (n = 42–64 cells in 2–3 experiments); ns, not significant.

Figure 5
Figure 5
PINK1 is required for parkin-dependent MDV formation

  1. Representative immunoblot of whole-cell lysates from HeLa cells transfected with non-targeting siRNA or siRNA targeting PINK1 (siPINK1), treated with 10 μM CCCP for 6 h in order to stabilize the PINK1 full-length band.

  2. HeLa cells transfected with GFP-parkin (green) and siRNA targeting PINK1 (siPINK1) or non-targeting control (ctrl siRNA), were treated with 25 μM antimycin A for 90 min, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). PDH E2/E3 bp-positive/TOM20-negative MDVs colocalizing with GFP-parkin (arrows) or not (circles) are indicated. Arrowheads indicate parkin-positive MDVs adjacent to mitochondria, possibly budding. Scale bar, 20 μm.

  3. Quantification of PDH E2/E3 bp-positive/TOM20-negative structures from (B); both the total number (white bars) and the number colocalizing with GFP-parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P-values are given first for GFP-/GFP-parkin-positive vesicles, then for total vesicle number (n = 42–60 cells in three experiments); ns, not significant; ***P < 0.001).

Source data are available online for this figure.
Figure 6
Figure 6
Parkin-dependent mitochondrial vesicle biogenesis is a rapid response triggered by ROS

  1. Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP-parkin cells treated with 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), and 20 μM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO-treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments).

  2. U2OS:GFP-parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 μm.

  3. Quantification of the total number of PDH E2/E3 bp-positive/TOM20-negative structures in U2OS:GFP-parkin cells observed at various times for the indicated treatment. Error bars represent the mean ± s.e.m. (n = 46–166 cells in 2–3 experiments).

  4. U2OS:GFP-parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp-positive/TOM20-negative structures colocalizing with GFP-parkin (green). Scale bars, 20 μm.

Source data are available online for this figure.
Figure 7
Figure 7
Cell-wide clearance of mitochondria via autophagy is triggered by depolarization and proceeds over a 24-h time period

  1. Representative immunoblot of whole-cell lysates from U2OS:GFP and GFP-parkin cells treated with DMSO, 25 μM antimycin A (anti A), 25 μM antimycin A with 10 μM oligomycin (anti A + oligo), or 20 μM CCCP for the indicated time period.

  2. Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from (A). Error bars represent the mean ± s.e.m. (n = 3 experiments).

  3. Quantification of mitochondrial clearance in U2OS:GFP-parkin cells treated as in (A), fixed and immunostained for TOM20. Data are shown as percentage of cells containing mitochondria, by TOM20 staining, visualized by fluorescence microscopy. Error bars represent the mean ± s.e.m. (n = 3 experiments, with at least 85 cells quantified per condition, per experiment).

  4. U2OS:GFP-parkin cells were treated as in (A) for the indicated time period, then fixed and immunostained for TOM20 (red). Cell boundaries are delineated in single-channel images. Scale bars, 20 μm.

Source data are available online for this figure.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Augustin S, Nolden M, Muller S, Hardt O, Arnold I, Langer T. Characterization of peptides released from mitochondria: evidence for constant proteolysis and peptide efflux. J Biol Chem. 2005;280:2691–2699. - PubMed
    1. d'Azzo A, Bongiovanni A, Nastasi T. E3 ubiquitin ligases as regulators of membrane protein trafficking and degradation. Traffic. 2005;6:429–441. - PubMed
    1. Bonifacino JS, Hurley JH. Retromer. Curr Opin Cell Biol. 2008;20:427–436. - PMC - PubMed
    1. Braschi E, Goyon V, Zunino R, Mohanty A, Xu L, McBride HM. Vps35 mediates vesicle transport between the mitochondria and peroxisomes. Curr Biol. 2010;20:1310–1315. - PubMed
    1. Campanella M, Casswell E, Chong S, Farah Z, Wieckowski MR, Abramov AY, Tinker A, Duchen MR. Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, IF1. Cell Metab. 2008;8:13–25. - PubMed

Publication types

MeSH terms

-