VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. Here, we demonstrate that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. VDAC1 deficient with polyubiquitination (VDAC1 Poly-KR) hampers mitophagy, but VDAC1 deficient with monoubiquitination (VDAC1 K274R) promotes apoptosis by augmenting the mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) channel. The transgenic flies expressing Drosophila Porin K273R, corresponding to human VDAC1 K274R, show Parkinson disease (PD)-related phenotypes including locomotive dysfunction and degenerated dopaminergic neurons, which are relieved by suppressing MCU and mitochondrial calcium uptake. To further confirm the relevance of our findings in PD, we identify a missense mutation of Parkin discovered in PD patients, T415N, which lacks the ability to induce VDAC1 monoubiquitination but still maintains polyubiquitination. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin-null flies. Taken together, our results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis.

The role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1; also called PARK5) in the pathogenesis of Parkinson’s disease (PD) has been controversial. Here, we find that the loss of UCHL1 destabilizes pyruvate kinase (PKM) and mitigates the PD-related phenotypes induced by PTEN-induced kinase 1 (PINK1) or Parkin loss-of-function mutations in Drosophila and mammalian cells. In UCHL1 knockout cells, cellular pyruvate production and ATP levels are diminished, and the activity of AMP–activated protein kinase (AMPK) is highly induced. Consequently, the activated AMPK promotes the mitophagy mediated by Unc-51–like kinase 1 (ULK1) and FUN14 domain–containing 1 (FUNDC1), which underlies the effects of UCHL1 deficiency in rescuing PD-related defects. Furthermore, we identify tripartite motif–containing 63 (TRIM63) as a previously unknown E3 ligase of PKM and demonstrate its antagonistic interaction with UCHL1 to regulate PD-related pathologies. These results suggest that UCHL1 is an integrative factor for connecting glycolysis and PD pathology.

Parkin is an E3 ligase that contains a ubiquitin-like (UBL)domain Parkinson disease (PD)3 is a neurodegenerative disorder that results from degenerated dopaminergic neurons in the substantia nigra. Most cases of PD are sporadic, and ϳ10% are familial (1). Gene mutations that are known to be responsible for the onset of the disease include PARK2 (Parkin), PARK6 (PINK1), PARK7 (DJ-1), LRRK2, and SNCA (␣-synuclein). LRRK2 and SNCA mutations are believed to cause PD through a gain of function, whereas PARK2, PARK6, and PARK7 mutations cause PD through a loss of function (2-4).Autosomal recessive early onset parkinsonism is linked to several loci, including PARK2 and PARK6 (5). The PARK2 gene encodes Parkin, an E3 ubiquitin ligase that consists of 465 amino acid residues. Parkin is composed of an ubiquitin-like (UBL) domain at the N terminus and a R1-in-between-ring (IBR)-Rind 2 (R2) motif at the C terminus (6 -8). Structurally, Parkin is a RING-type E3 ligase, but functionally it acts as a RING/HECT hybrid E3 ligase (9 -12). Parkin functions like a RING-type E3 ligase by interacting with E2 enzymes, UbcH7 and UbcH8, via the IBR domain, whereas the RING1 domain binds to substrates, allowing direct substrate ubiquitination. Parkin can also function like a HECT-type E3 ligase by catalyzing the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to the substrates via the active-site residues, Cys-431 and His-433. E2 enzymes that support Parkin function as a HECT-type E3 ligase are UbcH7, UbcH8, and Ubc13/Uev1a heterodimer (13,14).Substrates that are ubiquitinated by active Parkin include mitofusin (Mfn) 1 and 2, dynamin-related protein 1 (Drp1), voltage-dependent anion-selective channel protein 1 (VDAC1), mitochondrial Rho GTPase (Miro), and translocase of outer membrane 20 (TOM20) (15)(16)(17)(18)(19)(20). Parkin ligates these substrates with [21][22][23]. The substrates of Parkin with Lys-48-linked polyubiquitin chains are degraded by the ubiquitin proteasome system (23-26). However, those polyubiquitinated with Lys-63 or Lys-27 ubiquitin linkage recruit ubiquitin-binding adaptors such as histone deacetylase 6 (HDAC6) and p62/SQSTM1 (21,(27)(28)(29). The stability of Mfn1 and -2 and Drp1 are reduced by 30,31). TOM20 is both mono-and polyubiquitinated by Parkin by . In the case of VDAC1, Parkin catalyzes polyubiquitination with ubiquitin Lys-27 and Lys-63 linkages, which leads to recruitment of p62/SQSTM1 and subsequent induction of mitophagy (18,32).