Through controlling these mitochondrial proteins, the PINK1–Parkin pathway plays critical roles in maintaining mitochondrial homeostasis by regulating mitophagy, mitochondrial dynamics, and mitochondria-mediated apoptosis. Parkin is localized to the mitochondria and then ubiquitinates multiple substrates, including mitofusin1 (MFN1), mitofusin2 (MFN2), translocase of outer membrane20 (TOM20), mitochondrial Rho GTPase1 (Miro1), and voltage-dependent anion-selective channel 1 (VDAC1) ( 13– 15). For example, the mitochondrial depolarization resulting from carbonyl cyanide m-chlorophenyl hydrazine (CCCP) treatment accumulates PINK1 on the surface of damaged mitochondria and sequentially activates and translocates Parkin to mitochondria from the cytosol ( 10– 12). PINK1 is a serine/threonine kinase that is activated under mitochondrial damages. Parkin is phosphorylated at serine 65 located within the ubiquitin-like (UBL) domain by PINK1 and then its E3 ligase activity is activated ( 8, 9). Taken together, our results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis.Īmong the PD-associated genes mentioned above, PARK6 encodes PTEN-induced putative kinase1 (PINK1) and PARK2 encodes Parkin, which is a RING/HECT type E3 ubiquitin ligase. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin-null flies. 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. 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. 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. Here, we demonstrate that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. A model is presented which suggests that membrane potential plays a primary role in the proper orientation of the precursor signal sequence within the membrane, thus promoting processing and secretion.VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. These results suggest a role for membrane potential in the secretion of periplasmic proteins. Processing can be restored in CCCP-treated cells and in valinomycin-treated spheroplasts by dilution of the treated cells in fresh medium. Valinomycin, a potassium ionophore, also inhibits processing of the leucine-specific binding protein in spheroplasts. The levels of CCCP that inhibit processing also produce significant decreases in the membrane potential. The processing of both the leucine-specific binding protein and a plasmid-coded beta-lactamase is inhibited by phenethyl alcohol and by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). The leucine-specific binding protein of Escherichia coli is a periplasmic protein that is synthesized as a precursor and subsequently is processed during its secretion into the periplasmic space.