The kinase domain of mitochondrial PINK1 faces the cytoplasm

Abstract

Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD.parkin ͉ Parkinson's disease ͉ mitochondria ͉ topology P arkinson's disease (PD), the second most common neurodegenerative disorder, is a sporadic condition that can occasionally be inherited (1). The rationale for studying the rare genetic forms of PD is based on the phenotypic similarity between the familial and sporadic forms of the disease, implying that the two share important pathogenic mechanisms. Among the different gene products associated with familial PD (2), PTEN-induced putative kinase-1 (PINK1) is localized to the mitochondria (3-10), an organelle strongly linked to PD pathogenesis. Although recessively inherited PD mutations in PINK1 are found throughout the protein, they are most commonly found in the only recognized functional domain of PINK1 (3,11,12), which is a serine/threonine kinase domain similar to that in the Ca 2ϩ /calmodulin kinase family (13,14). Overexpression of wild-type PINK1 can rescue the phenotype caused by PINK1 mutations in Drosophila (15, 16), supporting the notion that the mutated allele gives rise to a loss-of-function phenotype.Human PINK1 is a 581-aa polypeptide with a predicted N-terminal mitochondrial targeting signal (MTS), consistent with the observation that PINK1 localizes to mitochondria (Fig. 1A) (3-10). Loss-of-function mutations in the gene encoding parkin (an E3 ubiquitin ligase) cause the most frequent forms of recessive familial PD (17). In Drosophila, parkin is thought to operate within the same molecular pathway as PINK1 to modulate mitochondrial morphology (15,16,18), an intriguing observation given the fact that parkin has been reported to essentially be cytosolic (19).Both the subcellular and submitochondrial locations of PINK1 and the authenticity of its reported substrates have been controversial. Although it is agreed that PINK1 is a mitochondrialtargeted protein, PINK1 has been reported to reside in the inner mitochondrial membrane (IMM) (6,7,9,20), the mitochondrial intermembrane space (IMS) (6,8,9), the outer mitochondrial membrane (OMM) (7), and even the cytoplasm (21-24). Furthermore, the tumor necrosis factor type 1 receptor-associated protein (TRAP1) and the serine protease HtrA2/Omi have been identified as putative PINK1 substrates (8, 9). Although TRAP1 is localized mainly i...

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

“…This study suggested that the N-terminus of PINK1 would be inserted in the OMM through the putative transmembrane domain (aa 94-110), with the kinase domain facing the cytosol. 13 Such a localization would argue against our hypothesis, as both N-and C-terminal domains of PINK1 are required to ensure a strong Beclin1 binding. Nevertheless, it is reasonable to speculate that a pool of PINK1-FL would not enter the OMM, but remain associated with mitochondria through anchoring proteins.…”

“…9,10 The full-length PINK1 (PINK1-FL) is processed within mitochondria to generate two mature proteins; 4,11 all three isoforms localize both to the mitochondria and cytosol, their relative ratio being regulated by several factors. [10][11][12][13] Increasing data have demonstrated that absence of functional PINK1 induces abnormalities of mitochondrial morphology. 6,14,15 In several studies (mostly in Drosophila), PINK1 was shown to promote fission acting upstream of the Fis1-Drp1 machinery, and the mitochondrial phenotype observed in PINK1 knockout flies or silenced cells was associated to reduced fission.…”

Drosophila Parkin requires PINK1 for mitochondrial translocation and ubiquitinates Mitofusin

“…PINK1's kinase activity, but not mitochondrial localization, appears to be necessary for Parkin translocation (23)(24)(25). Because PINK1 can be found extramitochondrially (27)(28)(29)(30) and may directly phosphorylate Parkin (31,32), this may be a mechanism to stimulate its translocation. Alternatively, it may phosphorylate a Parkin substrate, e.g., Mfn, and thereby provide a recruitment signal.…”

PINK1 as a Molecular Checkpoint in the Maintenance of Mitochondrial Function and Integrity

“…Determining the genetic link between Sir2 and PINK1 may provide a key for decoding the signaling mechanism between mitochondria and Sir2. Moreover, PINK1 faces the cytoplasm in the outer membrane (Zhou et al, 2008), and cytosolic PINK1 can protect neurons from MPTP (Haque et al, 2008). Therefore, PINK1 may directly phosphorylate and activate Sir2 in the cytoplasm.…”