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.

Fig. 1.

The kinase domain of mitochondrial PINK1 is accessible from the cytoplasm. (A) Schematic representation of PINK1 structure. The immunogens used to produce the antibodies (Abs) PINK1_175–250 and N4/15 are indicated by the red and blue lines, respectively. (B) SH-SY5Y cells expressing PINK1-IRES-eGFP stably are immunolabeled for a cytosolic protein (α-tubulin), a cytoplasm-facing OMM protein (TOM20), and an IMS protein (ENDOG) (red signals). eGFP is used as a general cell marker (green signals). PINK1 is labeled using PINK1_175-250 antibody. (Scale bar, 5 μm.)

Fig. 2.

PINK1 is a mitochondrial membrane integral protein whose kinase domain localizes at OMM. (A) Schematic representation of the submitochondrial compartment protein markers used in this study. (B) Mitochondria isolated from SH-SY5Y cells expressing PINK1 stably are subjected to carbonate extraction. Then, pellets (particulate fraction, P) and supernatants (soluble fraction, S) are analyzed by Western blotting using antibodies raised against PINK1 (PINK1_175–250), HSP60, ENDOG, peripheral membrane proteins (Cyto c), and membrane integral proteins (TOM20 and COX II). (C) Mitochondria isolated from SH-SY5Y cells expressing wild-type PINK1 stably are incubated with or without PK before analysis by immunoblotting using antibodies against PINK1 (PINK1_175–250), BCL-2, TOM20, TIM23, ENDOG, and HSP60.

Fig. 3.

Topology of endogenous PINK1 using PINK1_175–250 antibody. (A) mRNA levels of endogenous PINK1, parkin, and α-synuclein in SH-SY5Y cells and in PINK1 wild-type (WT) and knockout (KO) mouse brain. Total RNA extracted from each sample is quantified by real-time PCR (n = 3). Values represent means ± SD. PINK1 mRNA in KO mouse brain is below the limit of detection. (B) Using the antibody PINK1_175–250, there is no detectable signal for endogenous PINK1 in SH-SY5Y cells (vehicle control, left lane), whereas endogenous PINK1 is detected in the presence of the proteasome inhibitor MG132 (right lane). Western blot analysis of mitochondrial fractions shows an increased level of COX I with MG132 treatment. Accumulation of GFP^u, as shown by the increased GFP fluorescence intensity, indicates the successful inhibition of the proteasome pathway by MG132. (C) siRNA knockdown of PINK1. (Left) HeLa cells are cotransfected with either PINK1-HA/scramble control siRNA or PINK1-HA/PINK1 siRNA. Overexpressed full-length PINK1 and truncated ≈52-kDa PINK1 are diminished specifically by the PINK1 siRNA. (Right) Endogenous PINK1 is barely detected in whole HeLa cell lysates in the absence of MG-132, but it is clearly detected in the presence of MG-132. Endogenous PINK1 signals are markedly reduced by the PINK1 siRNA knockdown. (D) After MG132 treatment of SH-SY5Y cells, mitochondria are isolated and processed for PK protection assay as in Fig. 2.

Fig. 4.

The TM domain anchors PINK1 to the OMM by using PINK1_175–250 antibody. (A) Schematic representation of PINK1 constructs with various deletions. (B) SH-SY5Y cells transfected stably with empty vector (V), PINK1 (P), PINK1^Δ1–34, PINK1^Δ1–91, and PINK1^Δ1–150 are harvested to prepare cytosol and mitochondrial fractions. All preparations are analyzed by Western blotting. The percentages of various PINK1 constructs, including all degraded forms, which localize to mitochondria, are quantified by using Scion Image software and expressed as mean ± SD (n = 3). Note that for PINK1^Δ1–91, a large fraction of its cytosolic portion was degraded and for PINK1^Δ1–150 films are exposed for a shorter time to avoid saturation (thus the nonspecific band is not seen). Arrows, full-length constructs. Asterisks, nonspecific bands. (C) Mitochondria are isolated from SH-SY5Y cells expressing PINK1^Δ1–34 or PINK1^ΔTM stably followed by PK treatment as in Fig. 2. (D) Mitochondria are purified from SH-SY5Y cells expressing PINK1, PINK1^Δ1–77, or PINK1^ΔTM stably followed by permeabilization of the OMM with digitonin. After centrifugation, IMM (pellet, P) is separated from the OMM (supernatant, S). The purity of the fractions is assessed by probing the prepared fractions with antibodies to VDAC, TOM20, and COX II.