Parkin and PINK1 mediate Mitofusin ubiquitination and proteasomal degradation
Most known E3 Ub ligase substrates of Parkin have been identified in the cytosol, where Parkin normally localizes (Matsuda and Tanaka, 2010
). To identify potential Parkin substrates on mitochondria after depolarization and Parkin translocation, we examined the level of various mitochondrial proteins in the human neuroblastoma cell line SH-SY5Y, which expresses endogenous Parkin (Lutz et al., 2009
). 2 h after adding the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) to depolarize the mitochondria, we observed the selective reduction in expression of endogenous Mfn1 and Mfn2, human homologues of yeast Fzo1 that is known to be degraded by the proteasome (; Neutzner and Youle, 2005
). None of the other mitochondrial proteins examined displayed a reduction in protein levels, whereas Opa1 was cleaved as previously described (Ishihara et al., 2006
; Griparic et al., 2007
). Incubation of cells with a proteasome inhibitor, MG132, prevented the CCCP-induced decrease in Mfn1 and Mfn2 levels, which suggests that they are degraded by the proteasome. As the mitochondrial respiratory chain inhibitor rotenone has been used to generate an animal model of Parkinsonism (Betarbet et al., 2000
) and the oxidizing agent and herbicide paraquat has been linked to human Parkinsonism (Cochemé and Murphy, 2008
; Brooks et al., 1999
), we examined their effects on Mfn degradation. After a 24-h exposure to paraquat, SH-SY5Y cells display a clear decrease in levels of Mfn1 and 2 (). Rotenone treatment causes a more minor effect consistent with its weaker effect on Parkin translocation to the mitochondria (unpublished data). To specifically test the role of Parkin in mitofusin elimination, we compared Mfn levels in HeLa cells, which express little or no endogenous Parkin (Denison et al., 2003
; Pawlyk et al., 2003
), and in HeLa cells stably expressing YFP-Parkin (HeLa: YFP-Parkin). Endogenous Mfn1 and Mfn2 expression was not affected by YFP-Parkin in untreated cells, whereas their expression levels were greatly decreased after the addition of CCCP to HeLa: YFP-Parkin cells (). Furthermore, MG132 inhibited the elimination of Mfn1 and Mfn2, and also generated higher molecular weight bands of Mfn1, which is suggestive of ubiquitination (, MG132 plus lanes). Mfn levels were not reduced upon CCCP addition to HeLa cells not expressing YFP-Parkin. These data indicate that Parkin expression, when combined with mitochondrial uncoupling, induces mitofusin degradation via the Ub–proteasome system (UPS).
Figure 1. Mitochondrial depolarization induces selective degradation of mitofusins by Parkin. (a) Whole cell lysates of SH-SY5Y cells (30 µg of proteins) were subjected to SDS-PAGE after treatment with DMSO (control), CCCP (depolarized), or CCCP with a (more ...)
We compared the time course of YFP-Parkin translocation to mitochondria with that of mitofusin degradation after mitochondrial uncoupling. As shown in , 60 min after triggering mitochondrial depolarization with CCCP, the proportion of mitochondrial YFP-Parkin increased substantially, whereas mitofusin protein levels were drastically reduced. Both Mfn1 and Mfn2 are degraded in a Parkin-dependent manner, although Mfn1 degradation appears to be generally more robust than Mfn2 degradation. Interestingly, higher molecular weight bands of YFP-Parkin were also detected coincident with the time of mitochondrial translocation of Parkin. These high molecular weight Parkin bands were detected with YFP-Parkin and not with untagged Parkin, which is consistent with recent studies suggesting that the GFP portion of GFP-Parkin is a pseudo-substrate for Parkin (Matsuda et al., 2010
). This also suggests that the E3 ligase activity of Parkin is activated upon mitochondrial translocation. Simultaneous imaging of mCherry-Parkin and Mfn2-YFP expressed in HeLa cells after CCCP treatment also revealed a decrease in Mfn2-YFP signal, whereas untreated cells exhibited stable Mfn2-YFP intensity ( and Fig. S2 a
), which further indicated that CCCP-induced mitochondrial translocation of Parkin initiated degradation of mitofusin protein.
We also determined whether endogenous Mfn1 becomes ubiquitinated upon Parkin translocation to mitochondria. To this end, we applied denatured Mfn1 immunoprecipitation (IP) from control and CCCP-treated SH-SY5Y cells. The data show that CCCP treatment induces high molecular weight species of Mfn1 () that accumulated to even greater extents upon MG132 and CCCP cotreatment. Importantly, these high molecular weight species of Mfn1 cross-reacted with anti-Ub antibodies (, bottom). We also examined Mfn1 ubiquitination in HeLa cells with and without stable Parkin expression. As with SH-SY5Y cells, ubiquitinated Mfn1 was detected specifically after CCCP treatment in HeLa: YFP-Parkin cells, whereas no Mfn1 ubiquitination was detected after CCCP treatment of control HeLa cells ().
Figure 2. Mitofusin ubiquitination requires Parkin ligase activity. (a and b) SH-SY5Y (a), HeLa cells, or HeLa cells stably transfected with YFP-Parkin (b) were collected as cell lysates with denaturing buffer (see Materials and methods), then subjected to IP with (more ...)
Overall, these data indicate that Mfn1 is ubiquitinated and degraded in Parkin-dependent manner. To test this possibility further by knocking down PARK2
(Parkin) in SH-SY5Y cells, we examined whether Mfn degradation requires endogenous Parkin expression. We found that PARK2
knockdown inhibits Mfn degradation upon CCCP treatment (, PARK2
siRNA), which confirmed the essential role of Parkin in CCCP-induced Mfn degradation. We also determined whether pathogenic variants of Parkin affect the Mfn1/2 degradation process. To achieve this, we applied two Parkin mutants: ParkinR275W
, which translocates to depolarized mitochondria but fails to induce mitophagy; and ParkinC441R
, which fails to translocate to mitochondria (Narendra et al., 2010
). In contrast to transient expression of wild-type YFP-Parkin, expression of neither YFP-ParkinR275W
induced Mfn1/2 elimination upon CCCP treatment ().
PINK1, a mitochondrial kinase, is essential for the Parkin translocation to depolarized mitochondria and for Parkin-mediated mitophagy (Geisler et al., 2010
; Matsuda et al., 2010
; Narendra et al., 2010
; Vives-Bauza et al., 2010
). Therefore, we determined using PINK1
RNAi whether PINK1 is also required for CCCP-induced Mfn proteasomal degradation in the context of endogenous Parkin expression. The data show that RNAi-mediated depletion of PINK1
expression in Parkin-expressing M17 human neuroblastoma cells inhibited degradation of Mfn1 and Mfn2 ().
Figure 3. Mitofusin degradation requires PINK1 expression. (a) M17 cells expressing control or PINK1 shRNA were treated with CCCP or CCCP plus MG132. Lysates were immunoblotted with anti-Mfn1, Mfn2, PINK1, and Tim23. CCCP, 20 µM for 4 h; MG132, 30 µM, (more ...)
To further test the significance of Parkin activity for CCCP-induced Mfn degradation, we used an alternative strategy to inhibit endogenous Parkin activation. In contrast to inhibition of transcription, inhibition of translation by treatment with cycloheximide (CHX) prevents both the CCCP-dependent induction of PINK1 expression and Parkin translocation to CCCP-uncoupled mitochondria (Narendra et al., 2010
). Thus, we tested whether CHX would block Mfn1/2 degradation in CCCP-treated HeLa: YFP-Parkin cells and SHSY5Y cells. The data show that cotreatment of cells with CHX and CCCP completely prevents Mfn degradation (), which further supports the scenario in which Mfn ubiquitination and degradation not only requires endogenous PINK1 but also PINK1 accumulation and PINK1-dependent mitochondrial accumulation of Parkin during CCCP treatment.
To establish more directly that the ubiquitination of Mfn1 is mediated by Parkin, we reconstituted the ubiquitination process using a cell free system. As Mfn1 has two hydrophobic domains spanning the outer mitochondrial membrane (OMM), we used isolated mitochondria as a source of Mfn1 substrate to combine with purified recombinant maltose-binding protein (MBP)-tagged Parkin (MBP-Parkin) and purified UbcH7, an E2 Ub-conjugating enzyme found to function with Parkin (Matsuda et al., 2006
). Consistent with the in vivo data, we found that a high molecular weight ladder of Mfn1 appeared more prominent when the mitochondria were isolated from CCCP-treated HeLa cells, as compared with control HeLa cells (). Furthermore, high molecular species of Mfn1 were detectable in mitochondrial fractions from CCCP-treated HeLa cells incubated with wild-type MBP-Parkin, but not recombinant MBP-ParkinT415N
(), which is a Parkinson’s disease patient mutation lacking E3 ligase activity (Matsuda et al., 2006
). Using IP with the anti-Mfn1 antibody under denaturing conditions, we confirmed that these high molecular weight bands of Mfn1 were also detected with an anti-Ub antibody. Collectively, these results indicate that Parkin promotes ubiquitination of Mfn1 and that this ubiquitination can be recapitulated under in vitro conditions, which suggests a direct role of Parkin in ubiquitination of Mfn1. However, as mitochondria express at least one membrane-spanning E3 Ub ligase, MARCH5 (Karbowski et al., 2007
), Parkin could be activating another E3 ligase to ubiquitinate mitofusins. To determine whether MARCH5 is required for Parkin-dependent Mfn ubiquitination, we used wild-type HCT116 or MARCH5−/−
HCT116 cell lines that express abundant endogenous Parkin (Fig. S3 b
). After CCCP treatment for 3 h, Mfn1 and Mfn2 levels decreased comparably in HCT116 or HCT116 MARCH5−/−
cells (Fig. S3 b), which suggests that Parkin-induced Mfn degradation occurs in a MARCH5-independent manner.
Figure 4. Mitofusin ubiquitination requires Parkin and depolarization of mitochondria. (a) Mitochondrial fractions (30 µg) from HeLa cells treated with or without CCCP and recombinant MBP-Parkin wild-type or MBP-Parkin T415N proteins (left) were subjected (more ...)
We also asked whether Parkin might bind to Mfn1/2. The data show that in HeLa: YFP-Parkin cells, YFP-Parkin coimmunoprecipitated with endogenous Mfn1 and Mfn2, and that these interactions increased upon CCCP-induced depolarization of mitochondria (). In sum, molecular interactions of Parkin with Mfn1 and Mfn2 as well as cell free Parkin-dependent ubiquitination of Mfn proteins indicate that Parkin-mediated ubiquitination of Mfn1 and Mfn2 might be direct.
Role of p97 in Mfn proteasomal degradation
How membrane-spanning proteins such as the mitofusins are extracted from the OMM before their proteasomal degradation is currently unknown. We therefore examined if Mfn degradation requires the activity of p97, a AAA+ ATPase involved in the retrotranslocation of ER membrane-spanning proteins after ubiquitination and en route to proteasomal degradation (Ye et al., 2001
; Rabinovich et al., 2002
). To test this notion, we overexpressed wild-type p97 and the dominant-negative mutant p97 (E305Q/E578Q; p97QQ
; Ye et al., 2003
) in HeLa cells with or without transient Parkin expression. We found that Parkin-mediated Mfn1 ubiquitination was more apparent, and Mfn1 degradation was largely prevented by expression of the p97QQ
), which suggests that p97 mediates degradation of ubiquitinated Mfn1. Additionally, we found that overexpression of p97QQ
slowed down the turnover rate of Mfn1 (Fig. S4, c and d
) in control HeLa cells, which suggests that the UPS, through the p97 activity, might also mediate steady-state Mfn1 ubiquitination and degradation in the absence of mitochondrial uncoupling and Parkin expression.
Figure 5. AAA+ ATPase p97 mediates mitofusin degradation and accumulates on mitochondria after depolarization. (a) HeLa cells or HeLa cells transiently expressing Parkin were transfected with wild-type Myc-p97 or dominant-negative Myc-p97QQ (E305Q/E578Q). After (more ...)
To further test the role and mechanism of p97 in Parkin-dependent degradation of Mfn proteins, we examined the subcellular localization of p97 under conditions of inducing Parkin translocation to mitochondria. After 90 min of CCCP treatment, endogenous p97 accumulated on mitochondria in HeLa cells transiently expressing YFP-Parkin () but not in control HeLa cells or cells treated with tunicamycin, a stressor of the ER. The accumulation p97 on mitochondria upon Parkin translocation was confirmed by the data showing that Myc-p97 also translocated to the mitochondria in CCCP-treated Parkin-expressing cells. Interestingly, both wild-type Myc-p97 and Myc-p97QQ accumulated on mitochondria upon CCCP treatment in HeLa cells transiently expressing YFP-Parkin, but not on mitochondria in control HeLa cells lacking Parkin expression (). These data indicate that p97 accumulation on mitochondria requires Parkin expression and mitochondrial depolarization, and occurs independently of p97 ATPase activity.
Next, we examined whether PINK1 is required for mitochondrial accumulation of p97. In PINK1 knockout mouse embryonic fibroblasts (PINK1−/−MEFs), mitochondrial accumulation of p97 was not detectable upon CCCP treatment (), which indicates that p97 translocation is triggered by the activation of PINK1–Parkin activity upon depolarization of mitochondria. This was further supported by data showing that p97 accumulated specifically on uncoupled, Parkin-positive, and Ub-positive mitochondria ().
Figure 6. p97 recruitment to ubiquitinated mitochondria mediates PINK1–Parkin-induced mitochondrial elimination. (a) MEFs from PINK1+/+ or PINK1−/− mice were transiently transfected with YFP-Parkin and Myc-p97. Cells were treated with DMSO (more ...)
Mitochondrial fusion inhibition and Parkin-mediated mitophagy
, either promoting fission of mitochondria or suppressing fusion partially rescues the swollen mitochondrial phenotype of the pink1
mutant flies (Deng et al., 2008
; Poole et al., 2008
; Yang et al., 2008
; Park et al., 2009
). This suggests that the PINK1–Parkin pathway may normally promote fission of mitochondria. In addition, recent work indicates that mitochondrial fission is required for the progression of mitophagy (Nowikovsky et al., 2007
; Twig et al., 2008
; Kanki et al., 2009
), which suggests that promoting mitochondrial fission could compensate for a mitophagy deficit in Parkin-defective flies. Loss of mitofusins would be expected to inhibit mitochondrial fusion, perhaps fostering the autophagic elimination of depolarized mitochondria by segregating them from the mitochondrial network. We found that upon mitochondrial depolarization, Opa1 cleavage (Ishihara et al., 2006
; Griparic et al., 2007
) occurs more rapidly than mitofusin degradation (unpublished data), which suggests that the elimination of mitofusins does not cause the initial mitochondrial fragmentation but might delay fusion of mitochondria after uncoupler-induced fragmentation. To determine whether Parkin inhibits fusion of depolarized mitochondria independently of Opa1 cleavage, we examined the rate of recovery of tubular mitochondria after CCCP-induced depolarization and fragmentation of these organelles. Pretreatment with CCCP for 90 min generated depolarized, fragmented, Parkin-positive, and Mfn-negative mitochondria in HeLa: YFP-Parkin cells (, time 0). We then washed out the CCCP and analyzed the recovery of the tubular mitochondrial networks. After removing the CCCP, the recovery of mitochondrial networks in HeLa: YFP-Parkin cells was slower than in control HeLa cells (), despite the equal recovery of the longer form of Opa1 detectable both in the presence and absence of YFP-Parkin (). To further test the effect of Parkin on mitochondrial connectivity after CCCP washout phenotypes in , we applied a FRAP assay (Rizzuto et al., 1998
; Karbowski et al., 2007
). The fluorescence recovery of mitochondrial matrix-targeted YFP (mito-YFP) was analyzed in HeLa cells transiently expressing mCherry or mCherry-Parkin. The data show that at 90 min after CCCP washout, mCherry-expressing HeLa cells displayed a faster recovery of mito-YFP fluorescence compared with mCherry-Parkin–expressing cells ( [P = 0.0002, n
= 30]; and Fig. S5 a
). These results suggest that Mfn1/2 degradation promoted by Parkin maintains mitochondria fragmentation after mitochondrial depolarization. Parkin may therefore retain mitochondria in a fragmented state in order to facilitate mitophagy.
Figure 7. Parkin-promoted mitofusin degradation prevents refusion of damaged mitochondria with healthy mitochondria. (a) Scheme of washout design. HeLa cells or HeLa cells stably transfected with YFP-Parkin (green) were treated with CCCP for 90 min. (b and c) After (more ...)
Inhibition of mitochondrial fission hinders CCCP- and Parkin-dependent mitophagy
MEFs (Ishihara et al., 2009
) that display excessively fused mitochondria, we observed a noticeable decrease in Parkin-induced mitophagy compared with identically treated wild-type MEFs (, 24 h CCCP; and ), whereas YFP-Parkin recruitment () and Mfn elimination () were identical in these two cell types. To quantify mitochondrial interconnectivity in the DRP1−/−
MEFs, we applied FRAP analysis of mito-YFP as described in . As expected, mitochondria in DRP1−/−
MEFs transiently expressing mCherry-Parkin were more interconnected after CCCP treatment than in similarly treated wild-type MEFs (). These results indicate that inhibiting mitochondrial fission hinders Parkin-induced mitophagy. We also extended the results in DRP1−/−
MEFs to DRP1
knockdown HeLa cells. As in the DRP1−/−
knockdown HeLa cells transiently expressing YFP-Parkin and treated with CCCP displayed clumped mitochondria (, CCCP 24 h) and an 80% decrease in mitophagy () relative to control cells. Thus, in both DRP1−/−
MEFs and DRP1
knockdown cells, mitophagy is inhibited. This indicates that counteracting mitochondrial fission, which is a normal function of Mfn1/2, inhibits Parkin-mediated mitophagy.
Figure 8. Mitochondrial fission is required for Parkin-mediated mitophagy. (a) MEFs from DRP1+/+ or DRP1−/− mice were transiently transfected with YFP-Parkin. Cells were treated with CCCP and then immunostained with anti-Tom20. Cells expressing (more ...)
Collectively, we hypothesize that mitofusin degradation by mitochondria-associated Parkin inhibits the fusion of damaged mitochondria with healthy mitochondria, thereby segregating the impaired mitochondria from the network of fully functional mitochondria to facilitate their selective elimination by autophagy.