PD is one of several neurodegenerative diseases linked to mitochondrial dysfunction. The functions of specific mitochondrial proteins implicated in these diverse diseases include modifiers of respiratory chain complexes, metabolic enzymes, chaperones, and regulators of mitochondrial dynamics (24
). Here, we provide evidence that Pink1, which is genetically linked to PD, may participate in mitochondrial trafficking.
Milton and Miro are part of an essential protein complex that is known to link kinesin-1 to mitochondria for anterograde transport of mitochondria (18
). Milton (i.e., Milton-1 (OIP106) and Milton-2 (GRIF-1) in humans (26
)) serves as the adapter molecule that connects microtubules via kinesin to the mitochondrial-anchored Miro protein (20
). Miro (i.e., Miro-1 and Miro-2 in humans) is an atypical GTPase that contains tandem GTP-binding domains separated by a linker region with putative calcium-binding EF hand domains (15
). Genetic studies in yeast show that all domains of Gem1p (i.e., yeast Miro) are required for proper mitochondrial morphology (22
). Calcium stops mitochondrial movement, and it has been speculated that Miro's calcium-binding EF hand domains could be important for this effect (20
Taken together, these various published findings indicate that a function of the Miro/Milton complex in mitochondrial trafficking is well established. Thus, our identification of Pink1 as a member of this multi-protein complex makes it very likely that Pink1 is involved in the function of the complex, namely mitochondrial trafficking. To strengthen this finding, it is now critically important for the field to develop better Pink1 antibodies (14
) that will allow the detection and characterization of endogenous Pink1 protein complexes.
Pink1 expressed without its mitochondrial targeting sequence protects neurons against the dopaminergic neurotoxin MPTP (13
). These data strongly suggest that Pink1 can play a role relevant to PD mechanisms outside of mitochondria. Confocal immunofluorescence studies have suggested that ΔMTS-Pink1 partially overlaps with a mitochondrial marker stain (23
), although this result has been controversial (13
). Our study now provides data that help resolve this debate. First, we show by subcellular fractionation using differential centrifugation that about 20% of ΔMTS-Pink1 expressed in COS7 cells is found in the mitochondria-rich subcellular fraction. Our data is in agreement with the very recently published study showing that around 26% of exogenous Pink1 Δ1−91 in SH-SY5Y cells is mitochondrial (14
). The failure of Haque et al. (13
) to detect ΔMTS-Pink1 in the mitochondrial fraction using differential centrifugation could be explained by their use of NIH 3T3 cells. As they report, even wt Pink1 is only detectable at very low levels in the mitochondrial fraction of NIH 3T3 cells. Zhou et al. (14
) recently provided evidence that the kinase domain of mitochondrial Pink1 faces the cytosol. Their experiments suggest that Pink1 is anchored at the mitochondria by a transmembrane domain. Small amounts of full-length Pink1 have been reported in cytosolic fractions of overexpressing cells, suggesting that the full-length protein is not solely membrane-anchored (5
). Our new findings offer an alternative mechanism: that Pink1 66 and 55 kDa and ΔMTS all bind to the Miro-2/Milton-1 complex, which is well-known to reside at the outer mitochondrial membrane facing the cytosol. Indeed, we show that increasing either Miro or Milton retains more Pink1 in the mitochondria-rich fraction. Thus, we propose that Pink1 can also act in an MTS-independent fashion at the mitochondrion and can be retained on the surface of the mitochondrion by the Miro/Milton complex. Future experiments will determine whether Pink1 being part of the Miro/Milton complex is sufficient for its localization at the outer mitochondrial membrane and also whether the ΔMTS-Pink1/Miro/Milton complex is involved in the reported protective property of ΔMTS-Pink1 against toxicity by the mitochondrial toxin, MPTP.
Our mitochondrial morphology imaging data show increased mitochondrial fragmentation after Pink1 silencing that is fully suppressed by Miro overexpression. These data provide a functional link for mitochondria between Miro and Pink1. Although this rescue experiment apparently places the GTPase Miro downstream of Pink1, overexpression of Miro leads to an increased Pink1 66/55 kDa ratio, suggesting also potential upstream effects of Miro on Pink1. A similar phenomenon regarding Pink1 has been observed previously for Parkin (6
). The striking increase in mitochondrial fragmentation when Pink1 is silenced points to a defect in mitochondrial fusion. But one important concept in mitochondrial dynamics is that mitochondrial trafficking and fusion are not completely independent of one another (27
). Because proper mitochondrial fusion requires mitochondrial trafficking, it is possible that the observed fragmentation phenotype is mostly due to an impaired function of the Pink1/Miro/Milton protein complex. Future experiments will be necessary to confirm this hypothesis.
Alterations in the morphology of mitochondrial cristae have been found in Pink1 loss-of-function models (7
). In this regard, our identification of Mitofilin (also referred to as HMG or inner membrane mitochondrial protein) as a novel Pink1 binding protein may have special importance. Indeed, Mitofilin is known to be a critical organizer of mitochondrial cristae morphology and is indispensable for normal mitochondrial function (19
). The identification of Pink1 interacting proteins that are localized at different sites within mitochondria suggests multiple functional roles for Pink1. In order to better understand Pink1 in the context of pathogenic mechanisms of PD, it will therefore be important to analyze in more detail the Pink1/Miro/Milton protein complex in the context of the Pink1/Mitofilin interaction.