Running title: PCD8 transport
An alternative import pathway of AIF to the mitochondria
Shu-Fen Chianga,1, Chih-Yang Huanga,1, Shiow-Her Chioua,＊ and Kuan-Chih Chowb,＊
aGraduate Institute of Microbiology and Public Health, and bGraduate Institute of Biomedical
Sciences, National Chung Hsing University, Taichung, Taiwan
1These authors contributed equally to this work
Abstract: word counts, 131
Text: 15 pages, word counts: 4057 Figures: 6
＊Corresponding authors. Tel: +886-4-22840896 x 118; fax: +886-4-22859270. E-mail
address: [email protected], or Tel: 22840891 x 105; fax:
Abstract
In eukaryotic cells, transport of the newly synthesized proteins and phospholipids to the appropriate subcellular target compartments is essential for maintaining organelle
morphology and cell survival. In animal cells, mitochondria are major organelles containing DNA genome that encodes only for a small fraction of their proteins, which are required for the organelle function. Most mitochondrial proteins are encoded by the nuclear genes and imported to the mitochondria following protein synthesis. Programmed cell death protein 8 (PCD8), an essential FAD-dependent NADH oxidase for the oxidative phosphorylation, is located in the intermembranous space and contains mitochondrial localization signals.
However, the import mechanism of PCD8 to the mitochondria is not well illustrated. Here we show that PCD8 is imported from the endoplasmic reticulum to the mitochondria via
mitochondria-associated membranes (MAM) and the transport vesicles.
Key words: ATAD3A, DRP1, endoplasmic reticulum, Mfn-2, mitochondria,
mitochondria-associated membrane, programmed cell death protein 8, transport vesicles
Abbreviations used are: PCD8, apoptosis inducing factor 8; ATAD3A, ATPase family, AAA domain containing 3A; DRP1, dynamin-related protein 1; ER, endoplasmic reticulum; MAM, mitochondria-associated membrane; Mfn-2, mitofusin-2; MLS, mitochondrial localization signal; MOM, mitochondrial outer membrane; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PM, post-mitochondrial; PS, phosphatidylserine; TIM, translocase of mitochondrial inner membrane; TOM, translocase of mitochondrial outer membrane; TV, transport vesicle
1. Introduction
Mitochondria are major organelles that have their own DNA genomes. However, their genomes encode only for thirteen proteins that are part of the oxidative phosphorylation. Most of the estimated 1,500 human proteins, which are essential for the mitochondrial function, are nuclear encoded. It is believed that these proteins are synthesized in the cytosolic ribosomes as precursor proteins or co-translational proteins, and imported into mitochondria via mitochondrial localization signal (MLS) and the translocase of
mitochondrial outer membrane (TOM) complex[1, 2]. The precursor proteins are further
guided by chaperones to the receptor of TOM complex and imported into mitochondria [3, 4]. The co-translational proteins are synthesized on mitochondria-associated ribosomes, forming nascent polypeptide-associated complex (NAC) on the surface of mitochondria, and directly imported into mitochondria [1, 5]. However, several mitochondrial proteins do not contain the evident MLS and they are located on the outer membrane (MOM), in the
intermembranous space (IMS), on the inner membrane (MIM) or in the matrix, e.g., mitofusin 2 (Mfn-2), cytochrome c, optic atrophy 1 (OPA1) and cytochrome P450 family 11A1 (cyp11a1), respectively [6-8].
Programmed cell death protein 8 (PCD8), also called apoptosis-inducing factor (AIF), is a FAD-dependent NADH oxidase, which is located in the intermembranous space of
mitochondria and has been suggested playing an important role in the oxidative
phosphorylation of mitochondrial complex I and III [9]. The full-length PCD8 contains 2 potential mitochondria localization signals (MLS) at the N-terminus, and 2 putative nuclear localization signals (NLS) in the middle of the protein [10]. Interestingly, programmed cell death protein 8 and cytochrome c are all located in IMS, but presence of free cytochrome c and PCD8 in cytoplasm is hazardous to cells. Following genotoxic challenges, PCD8 and cytochrome c are released from the mitochondria [11-13]. PCD8 is further translocated to the
nucleus to trigger DNA fragmentation and initiation of apoptosis [10, 13, 14]. Cytochrome c, on the other hand, stays in the cytoplasm and activates apoptotic peptidase activating factor 1 (Apaf1) to cleave procaspase 9, which then initiates the caspase cascade and commits cells to apoptosis [11, 15]. To protect Apaf1 from an inadvertent activation and to prevent cells from unprogrammed apoptosis, the newly synthesized cytochrome c and PCD8 have to be
protected by charperons or be rapidly concealed before transporting to the mitochondria. However, the mechanism of importing PCD8 to the mitochondria is not well illustrated.
Recently, when studying expression of dynamin-related protein 1 (DRP1), mitofusin-2 (Mfn-2), ATPase family, AAA domain containing 3A (ATAD3A) and in lung cancer cells, we found that silencing of DRP1 increased bulging of the endoplasmic reticulum (ER) and mitochondria-associated membrane (MAM) [16-19]. Silencing of ATAD3A, on the other hand, increased mitochondrial fragmentation and the number of vesicles around the dilated ER or MAM [20]. Silencing of Mfn-2 increased mitochondrial fragmentation and the number of vesicles around the mitochondria. Based on those results, we hypothesized that there could be an alternative import pathway for some of the mitochondrial proteins, and that at least three proteins, i.e., DRP1, ATAD3A and Mfn-2, were required for the protein trafficking. In this report, our data show that some of mitochondrial proteins, such as PCD8 and cytochrome c, are indeed synthesized and packaged in the ER, en route through mitochondria-associated membrane and transported via vesicles to the mitochondria before fusing with the
2. Experimental procedures
2.1. Cell lines
Three human cancer cell lines, including 2 lung adenocarcinoma cell lines (H23 and H838) [20, 21], a uterine cervical cancer cells (HeLa), and were used in the study. Cells were maintained at 37C as a monolayer in RPMI 1640 supplemented with 10% fetal calf serum, 100 g/ml of streptomycin and 100 IU/ml of penicillin. To generate cells that stayed at G1/G0 phase of cell cycle, H23 cells were grown to 70% confluence from the initial seeding and changed to medium containing 0.5% of serum for 48 hours. For HeLa cells to be at the different phases of cell cycle, the cells were respectively treated by serum starvation, double thymidine block or thymidine-nocodazole. To enrich cells at S phase, cell cycle progression of Hela cells were blocked with 2 mM thymidine for 18 hr, before a release for 9 hr. The cells were then treated with a second thymidine block for 15 hr and a release for 6 hr. The enriched G2/M cells were obtained by thymidine-nocodazole treatment. The cell cycle was blocked with 2 mM thymidine for 24 hr, a release for 3 hr, and incubated with 100 ng/ml nocodazole for 12 hr. The cells were collected and stained with propidium iodide before analysis by a
flow cytometry (Beckman Coulter CytomicsTM FC500).
2.2. Fractionation of cellular components
Fractionation of subcellular components was performed according to the instruction manuals of Calbiochem (http://www.merckbiosciences.co.uk) with minor modifications. The cells were detached from culture plates by treating with dissociation buffer (Sigma, St Louis, MO) at 37°C for 2-5 minutes. After washing with PBS, the cells were re-suspended in H
buffer (10 mM Tris-HCl, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM
Na-EGTA, 1 mM dithiothreitol, 250 mM sucrose, 0.1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 10 μg/ml trypsin inhibitor) at 4°C for 15 min. After 80 strokes with a B pestle
in a Douncer homogenizer, the unbroken cells were removed by centrifugation at 30 g for 5 min. The nuclei were collected by spinning the solution at 80 g for 10 min, and
mitochondria were collected from the supernatant by centrifugation at 6,000 g for 20 min. Following centrifugation at 20,000 g for 20 min to remove the insoluble debris, the final supernatant was used as post-mitochondrial fraction [20]. For isolating MAM and
microsomes, the homogenates were centrifuged twice at 600 g to completely remove nuclei. The supernatant was centrifuged at 10,500 g to separate the crude microsomal (microsomes and cytosol) from the crude mitochondrial (MAM and mitochondria) fractions. The crude microsomal fractions (supernatant) were subjected to an ultracentrifugation at 100,000 g for 60 min at 4°C to pellet the microsomes, while the supernatant was used as cytosol. The crude mitochondrial fractions (pellet) were resuspended in 300 μl of ice-cold mannitol buffer A (0.25 M mannitol, 5 mM HEPES, 0.5 mM EGTA, pH 7.2) and layered on top of 10 ml of a 30% Percoll suspension in mannitol buffer B (0.25 M mannitol, 25 mM HEPES, 1 mM EGTA, pH 7.2). Mitochondria and MAM fractions were separated during the formation of self-generating Percoll gradient by ultracentrifugation at 95,000 g for 65 min at 4°C. Both isolated fractions were diluted five times in sucrose homogenization medium and subjected separately to a centrifugation at 6,300 g for 10 min at 4°C. The pellet was used as the purified mitochondria, while the supernatant was further separated by
centrifugation at 100,000 × g for 30 min at 4°C, and the pellet was used as the purified MAM fraction. The ultracentrifugation was performed using the Beckman SW41 rotor, and all of the fractions were resuspended in sucrose homogenization medium before immunoblotting analysis.
Procedure for immunoblotting has been described previously [21]. Briefly, proteins were separated in a 10% polyacrylamide gel with 4.5% stacking. After electrophoresis, proteins were transferred to a nitrocellulose membrane. The membrane was then probed with specific antibodies. The signal was amplified by biotin-labeled goat anti-mouse IgG, and peroxidase-conjugated streptavidin. The protein was visualized by exposing the membrane to an X-Omat film (Eastman Kodak, Rochester, NY) with enhanced chemiluminescent reagent (NEN, Boston, MA). Following characterization with immunoblotting, the same antibodies were used for immunofluorescence confocal microscopy. For the staining, cellular uptake of MitoTracker® green FM (Molecular Probes, Inc., Eugene, OR) was used to label
mitochondria. The cells were then fixed with 4% formaldehyde at room temperature for 15 minutes. After washing three times with PBS, cells were incubated with the primary
antibodies for 90 minutes, and washed three times with PBS. The secondary antibodies used were rhodamine (TRITC)-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Nuclei were stained with
4',6-Diamidino-2-phenylindole (DAPI). The slide was examined and the images were captured in a laser scanning confocal microscope (LSM510, Zeiss, Chicago, IL). The images were processed by a Zeiss LSM Image Browser (LSM5 Image Software, Zeiss, Chicago, IL) and a Photoshp 7.0 software (Adobe Systems Incorporated).
2.4. Construction of PCD8–expressing plasmids
A forward and a reverse primer sequences for cloning expression vector containing the full-length PCD8 (AF100928, coding sequence, nts 179-2020) are listed in the following: 5’-TTGGAATTCATTATGTTCCGGTGTGGAGGCCTG-3’ (nts 43-63, Eco RI site is underlined and the initiation codon is in bald-phase letters); and
in italic and underlined and the stop codon is in bald-phase letters). The amplified DNA fragments were digested with the respective restriction enzymes and then inserted into the vector pcDNA3.1.
2.5. Electron microscopy
Electron microscopy was carried out using a routine protocol [20]. Briefly, cells were fixed with 2.5% glutaraldehyde (EM grade, Sigma, St Louis, MO, USA) in 100 mM phosphate buffer (PB, pH 7.2), incubated at 4°C overnight. The cells were washed with PB three times before post-fixation with 1% osmium tetroxide in PB for two hours. After
removal of the fixative with distilled water, the cells were suspended in 2% molten agar. The agar blocks were trimmed and dehydrated in a serial dilution of ethanol for 15 minutes each. The blocks were further dehydrated with 100% ethanol for 15 minutes three times, and infiltrated with 100% ethanol/LR white (1:1) mixture overnight. The blocks were changed to the pure LR white (Agar Scientific Ltd., Essex, UK) and continued infiltration at 4°C for 24 hours, before transferred to a capsule filled with LR white, which were polymerized and solidified at 60°C for 48 hours. The resin blocks were trimmed and cut with ultramicrotome (Leica Ultracut R, Leica Mikrosysteme GmbH, Vienna, Austria). The thin sections were transferred to 200 mesh copper grids, and stained with 2% uranyl acetate for 30 min, and 2.66% lead citrate (pH 12.0) for 10 min, before observation with an electron microscope (JEM1400, JEOL USA, Inc., Peabody, MA, USA) at 100-120 kV. For gene silencing experiments, cells were harvested 48 hours following siRNA treatment.
2.6. Post-embedded immune-gold procedure for electron microscopy
Following fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS buffer at 4°C for 18 hr, the cells were washed with PBS 3 times. The cells were then permeabilized
with 0.2% Triton X-100 in PBS for 20 min. After washed with PBS for 3 times, the cells were embedded in 2% agarose and the agarose block was cut into several pieces prior to dehydration. After dehydration in a series of ethanol with escalated concentration, samples were embedded in LR White resin. Thin sections were cut by ultramicrotome and transferred to nickel grids. The non-specific binding sites on the thin sections were blocked with 50 mM glycine and 1% BSA in PBS for 15 min. The sections were incubated with the primary
antibody (1:50) in incubation buffer (0.1% BSA and 15 mM NaN3 in PBS) for 2 hr at room
temperature. The antibodies were then removed by washing the grids with incubation buffer for 6 times and the grids were further incubated with 15 nm gold-conjugate secondary
antibody (1:25) (Aurion, Wageningen, Netherlands). Following removal of the non-binding
immune-gold by repeated washing of the grids with incubation buffer, the sections were post-fixed with 2% glutaraldehyde in PBS for 15 min and stained with uranyl acetate and lead citrate before observation with an electron microscope.
3. Results
3.1. Newly expressed PCD8, of which the levels increase during the S and G2 phases of cell cycle, is resistant to trypsin, and is detected not only in mitochondria
The biogenesis of mitochondria is in S-phase [22]. We therefore anticipated that biosynthesis of PCD8 was cell cycle-dependent. Serum-starved quiescent H23 cells, when replenished with fresh serum, synchronously entered cell cycle progression [23]. Levels of PCD8 increased about 10 hours after serum stimulation, and reached a peak at 20 hours (Fig. 1A). The results were verified by an analysis of flow cytometry, in which PCD8 levels increased in the S and G2 phases of cell cycle (Fig. 1B, 1C, 1D).
Several mitochondria-associated proteins, e.g., dynamin-related protein (DRP) 1, and the ATPase family, AAA domain containing 3A (ATAD3A), also increased during these phases (Fig. 1C) [20, 21]. Interestingly, in addition to the mitochondrial fractions, PCD8 was also detected in post-mitochondrial fractions (Fig. 2A), and PCD8 from these two fractions was resistant to trypsin in synchronized S and G2 cells. However, the PCD8 became sensitive to trypsin when the post-mitochondrial fractions were pre-treated with digitonin (Fig. 2A), suggesting that the PCD8 in S and G2 phases could be protected by digitonin-sensitive molecules, e.g., phospholipids. In general, the mitochondrial proteins are believed to be imported into mitochondria via TOM40 complex. Deficiency of TOM40 components will reduce protein levels of the mitochondria. However, we did not detect the evident reduction of PCD8 in the total cell lysate or mitochondrial fractions when expression of TOM40 channel or TOM22 receptor was silenced (Fig. 2B and 2C), suggesting that PCD8 might not be imported through the TOM40 translocator of the mitochondria. These results in part supported our hypothesis that PCD8 might not be imported to mitochondria via the routine TOM passage, and in part implicated that there might be an alternative protein transport pathway to mitochondria.
3.2. Subcellular localization of PCD8 as determined by immunoblotting
Because results of trypsin digestion suggested that PCD8 could be present within the phospholipids, we then used sucrose gradient ultracentrifugation and immunoblotting to determine in what membrane fractions of lung cancer cells the PCD8 was existent. As shown in Fig. 3, PCD8 and 78-kDa glucose-regulated protein (GRP78) were detected in the ER [the light membrane (LM) fraction], the mitochondria-associated membrane (MAM) and the mitochondrial fractions of the wild-type H838 cells, but not in the cytosol. Knockdown of
80-kDa DRP1 (DRP1kd) expression, the essential GTPase for budding off of transport vesicles
from the MAM, decreased levels of PCD8, ATAD3A, GRP78, mitofusin (Mfn)-1 and Mfn-2 in the fractions of mitochondria, the target organelle of these proteins (Fig. 3, the middle panel). However, levels of these proteins increased in the fractions of MAM, suggesting that expression of DRP1 could influence the transport of these proteins from the ER to
mitochondria, and that the MAM could be a docking area for the shipment of these cargo
proteins to the mitochondria. Knockdown of Mfn-2 (Mfn-2kd) expression, on the other hand,
decreased levels of Mfn-1 and GRP78 in the mitochondrial fractions (Fig. 3, the right panel), indicating that Mfn-2 was essential for the fusion of transport vesicles into mitochondria. These results clearly showed that knockdown of DRP1 or Mfn-2 could interfere PCD8 distribution in the ER, the MAM and the mitochondria. Silencing of import-related protein expression would leave the PCD8, the cargo proteins, build up in the prior membrane fractions, i.e., the ER, the MAM and the transport vesicles.
3.3. Subcellular localization of PCD8 as determined by confocal microscopy
To further verify our hypothesis, we used confocal immunofluorescence microscopy to examine the distribution pattern of full-length recombinant human PCD8 in cells. As
anticipated, rhPCD8 was mainly localized in the mitochondria (mitochondria were visualized
by live dye of mitochondria, DsRed-Mito; Fig. 4A, upper panel). In DRP1kd cells, some of
rhPCD8 did not co-localize with mitochondria (Fig. 4A, central panel), but with the ER and MAM (ER was visualized by ER-retention signal KDEL-conjugated GFP; Fig. 4B, central
panel). In ATAD3Akd cells, the rhPCD8 was mainly present in the cytoplasmic vesicles (Fig.
4, A and B, lower panel).
To further differentiate whether the co-localization was in the ER or MAM, we labeled the MAM with human phosphotidylserine synthase 1 (huPSS1)-conjugated GFP. In the resting cells, no evident co-localization was detected between rhPCD8 and the MAM (Fig. 4C, upper panel). In replicating cells, few areas of co-localization between rhPCD8 and the
MAM were detected (Fig. 4C, lower panel). In DRP1kd H838 cells, co-localization between
the MAM and rhPCD8 became evident (Fig. 4D, central panel), suggesting that inhibition of budding off of transport vesicles from the MAM accumulated cargo proteins, such as PCD8,
in the docking mitochondria-associated membrane. In ATAD3Akd cells, owing to the lack of
moving ability, the transport vesicles which had been budded off from the MAM would be dispersed around the MAM.
We then examine whether the same condition could be observed for cytochrome c, a mitochondrial protein that was also located in the intermembranous space. Compared to the wild-type cells (Fig. 4E, upper panel), silencing of DRP1 expression increased levels of cytochrome c in the bulging subcellular structures (Fig. 4E, central panel). Knockdown of ATAD3A expression, however, increased levels of cytochrome c in the cytoplasmic vesicles (Fig. 4E, lower panel). The bulging structure was identified as mitochondria-associated membrane (Fig. 4F). When organelle marker was changed to ER-retention signal KDEL-conjugated GFP (Fig. 4G, upper panel), the bulging cytochrome c-positive figures became distinct from the ER (Fig. 4G, central panel), indicating that the bulging figures was the
mitochondria-associated membrane, which could be a specialized part of the ER. These data are consistent with those of rhPCD8. Our previous results showed that ATAD3A acted as a critical ATPase for moving transport vesicles from the MAM to the mitochondria. Silencing of ATAD3A expression would therefore increase levels of cytochrome c in the cytoplasmic vesicles (Fig. 4G, lower panel), further supporting our hypothesis that some of mitochondrial proteins were imported from the ER and the MAM [20].
3.4. Subcellular localization of PCD8 as determined by electron microscopy
For more detailed study, lectron microscopy was employed to determine the ultrasubcellular localization of PCD8. The PCD8-specific immunogold particles were identified in the cytoplasm, the ER, the transport vesicles, and the mitochondria (Fig. 5, A
and B). As noted above, knockdown of DRP1 (DRP1kd) expression increased the average
length of mitochondria and the bulging ER (or mitochondria-associated membrane, MAM). Ultrastructurally, PCD8 accumulated in the dilated ER/MAM (Fig. 5, C and D). Interestingly,
in ATAD3Akd cells, the PCD8 signal was more evident in the transport vesicles (Fig. 5, E and
F). In Mfn-2kd cells, however, the PCD8 signal was mainly present in the transport vesicles
around the mitochondria (Fig. 5, G and H), suggesting that other than mitochondrial fusion, Mfn-2 also played a role in maintaining the ER integrity and probably in the fusion of transport vesicles into mitochondria. These results confirmed our hypothesis that DRP1, ATAD3A, and Mfn-2 were essential for maintaining shaping of the ER and the mitochondria, in particular in the shipment of transport vesicles, which were budding off from the
4. Discussion
The results presented above support our hypothesis that PCD8 is imported from the ER to the mitochondria, probably via mitochondria-associated membranes (MAM) and transport vesicles. This process requires at least three proteins: DRP1, ATAD3A and Mfn-2. DRP1 is important for the formation of transport vesicles from the mitochondria-associated
membranes, while ATAD3A is essential for transporting vesicles to the mitochondria, and Mfn-2 is critical for the fusion of transport vesicles into the mitochondrial outer membrane. A simplified sketch of this mechanism is shown in Fig. 5. In this case, cells can import PCD8 as well as other proteins and phospholipids from the ER to the mitochondria.
Our previous study had evidently demonstrated such possibility, by using that antibodies specific to ATAD3A immunologically precipitated PCD8 in a detergent-free
post-mitochondrial fractions, but not in a cytosolic fractions. Moreover, antibodies specific to ATAD3A also precipitated eukaryotic elongation factor-2 (eEF-2), Mfn-2, DRP1 and OPA1, indicating that these molecules were interacting with one another in the post-mitochondrial fractions [13]. On the other hand, antibodies specific to PCD8 only precipitated PCD8 in a cell lysate containing nonionic detergent NP-40, but not in detergent-free post-mitochondrial fractions (data not shown), further suggesting that PCD8 was prevented from binding to antibodies by NP-40 sensitive molecules, such as phospholipids.
Interestingly, mitochondria do not synthesize phospholipids de novo. Essential
phospholipid, e.g., phosphatidylserine (PS), is synthesized in the ER and then transported to the mitochondria before converting to phosphatidylethanolamine (PE). An elegant study by Shiao et al. showed that phospholipid exchange between the ER and the mitochondria was protein-dependent [24]. They suggested that phospholipid exchange might possibly be via mitochondria-associated membranes, an specialized area of the ER which was closely contacted with the mitochondria, and had been implicated in regulating phospholipid
biosynthesis and transport, calcium transmission, energy metabolism and cell survival [25-27] By studying biosynthesis of phosphatidylcholine (PC), de Kroon et al. showed that phosphalipid import required both ATP and GTP [28]. In addition, DRP1 was not only detected on the ER and the mitochondria, but also on a number of small cytoplasmic vesicles
[19]. Expression of a dominant negative DRP1K38A gene markedly reduced the number of
cytoplasmic vesicles and induced bulging of the mitochondria-associated membrane (MAM), supporting our data that proteins and phospholipids formed cargo vesicles in the MAM [18-20, 29, 30]. It is worth noting that biogenesis of mitochondria is at the S-phase. Expression of many mitochondrial proteins, e.g., cytochrome c oxidases, is also cell cycle-dependent and regulated by nuclear respiratory factor (Nrf) [31, 32]. Unless these proteins were
continuously synthesized in the cytoplasm; otherwise, they would swarm the mitochondrial periphery and aggregate at the TOM/TIM complexes.
Lately, Mfn-2 was identified as a linker protein on the MAM and the mitochondria, which tethered MAM and mitochondria together [17]. In addition to mitochondrial fusion, Mfn-2
was imperative for the regulation of Ca2+ flow between the ER and mitochondria [16, 33].
However, besides a cleavage site for mitochondrial presequence and two peroxisomal targeting signals, no evident mitochondrial targeting sequence was detected in the Mfn-2 protein (Supplementary Figure S1). Because peroxisome is derived from the ER, and formation of peroxisome is via MAM, which requires DRP1 and AAA-ATPases (Pex1 and Pex6) [34, 35], findings by de Brito and Scorrano indicate that targeting of Mfn-2 to the ER and mitochondria is either mediated by a yet to be determined mechanism or the import of some mitochondrial proteins is by an alternative route [20, 36], which conceivably is via the MAM and transport vesicles.
By studying hepatitis c virus core protein (HCVCP), Schwer et al. showed that HCVCP was frequently detected in the ER, the light membrane, MAM and mitochondria. By
investigating intracellular trafficking of human cytomegarovirus (hCMV) UL37 proteins, Bozidis et al. found that the full-length UL37 protein (gpUL37) and the derivatives were all identified in the MAM and sometimes in mitochondria [37]. Elegant studies by Sun et al. and Burikhanov et al. showed that GRP78, a chaperone protein of ER, was also detected in mitochondria, nucleus and plasma membrane [38, 39]. These results considered together with our current data clearly suggest that sorting of some mitochondrial proteins may take an alternative route, from the ER and the mitochondria-associated membrane. In this way, phospholipids and proteins could be simultaneously transported from the ER to mitochondria without causing imbalance between the quantity of phospholipids and proteins in target organelles. Our hypothesis provides a reasonable explanation for why silencing of any of the three genes, DRP1, ATAD3A or Mfn-2, concomitantly alters morphology of the ER and mitochondria, and in severe cases it induces autophagy. In conclusion, we find the passage for PCD8 transport from the ER to mitochondria. This process requires at least three proteins, DRP1, ATAD3A and Mfn-2, which have respective roles: DRP1 is for forming transport vesicles from the MAM, ATAD3A is for shipping the transport vesicles to the mitochondria, and Mfn-2 is for the fusion of transport vesicles into the mitochondria (Fig. 6). It should be noted, however, that other proteins could be involved in this process [36]. In an ongoing study, we are investigating how many mitochondrial proteins are imported via this route.
Acknowledgements
We thank Dr. F. Peruzzi (Department of Neuroscience and Center for Neurovirology, Temple
University School of Medicine, USA) for DsRed-mito; Dr. D. J. Snyders (Department of
Biomedical Sciences, University of Antwerp, Belgium) for DsRed-ER; Drs. Jiuping Ding and Tao Xu (Key Laboratory of Molecular Biophysics, Huazhong University of Science and Technology, People Republic of China) for pEGFP-ER and Dr. Anamaris M. Colberg-Poley
(Center for Cancer and Immunology Research, Children's Research Institute, Children's
National Medical Center, USA) for mEGFP-huPSS-1. The authors declare that they have no competing interests in this study.
Funding
This study was supported by the Comprehensive Academic Promotion Projects (NCHU 995002, Ministry of Education, Executive Yuan, Taiwan, to K.C. Chow).
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Figure legends
Fig. 1. Expression level of PCD8 increased during proliferation phase of cell cycle
progression. A, Expression levels of PCD8 as measured by immunoblotting. Whole cell
lysate (5 × 106 cells per lane) was prepared from quiescent H23 cells (Time 0) and
serum-stimulated cells at five-hour interval for 25 hours. Expression ratio of PCD8, which was measured by density scanning of the exposed films, is shown in the lower panel. B, Using a flow cytometry to analyze cell cycle progression of H23 cells, which were synchronized by serum starvation for 48 hrs and then released by addition of fresh serum. The harvest intervals were 5 hr. C, Expression level of PCD8 at the different phases of cell cycle
progression in HeLa cells. G1phase cells were collected following serum starvation for 48 hr, S phase cells were collected 6 hr following release from double thymidine block, and G2/M phase cells were collected from thymidine-nocodazole treatment. D, Using a flow cytometry to analyze cell cycle progression of HeLa cells, which were harvested from the control, serum starvation, double thymidine block and release, and thymidine-nocodazole co-treatment.
Fig. 2. PCD8 is protected by digitonin-sensitive molecules and is not imported into
mitochondria through TOM complexes. A, Protein was collected from mitochondrial (Mito) and post-mitochondrial (post-mito) fractions at S phase, and PCD8 from both of these two fractions was resistant to trypsin (in 0% digitonin). When these fractions were pre-treated with 0.2% of digitonin, the PCD8 became sensitive to trypsin, suggesting that in S phase PCD8 was protected by trypsin-resistant molecules. B, Immunoblot analysis of total cell
lysates which were from the wild-type, TOM20kd, TOM22kd, and TOM40kd H838 cells. C,
Immunoblot analysis of mitochondrial fractions which were from the wild-type, TOM22kd
Fig. 3. Immunoblot analysis of mitochondria-associated proteins in subcellular fractions.
Immunoblot analysis of membrane fractions isolated by sucrose gradient ultracentrifugation
in the wild-type (WT, the left panel), the DRP1kd (the central panel) or Mfn-2kd (the right
panel) human lung cancer H838 cells. WT, wild-type; Cyto, cytosolic fraction; LM, light membrane; MAM, mitochondria-associated membrane; Mito, mitochondrial fraction. Cytochrome oxidase IV (COX IV) is a mitochondrial marker
Fig. 4. The effect of mitochondrial import-related protein expression on the subcellular
distribution of PCD8 as determined by confocal immunofluorescence microscopy. A,
Compared to the wild-type H838 cells, knockdown of DRP1 (DRP1kd, the central row) or
ATAD3A expression increased cytoplasmic level of recombinant human PCD8 (rhPCD8) (arrows, green fluorescence). Mitochondria were stained with DsRed. The images were processed by a Zeiss LSM Image Browser (LSM5 Image Software, Zeiss, Chicago, IL). R = green fluorescence/red fluorescence; if R = 1, which means that the intensity of green fluorescence equals to that of the red fluorescence, and that the green fluorescence overlaps with the red fluorescence. The overlapped fluorescence which appears on the Figure is yellow. If R > 1, which means that the intensity of the green fluorescence is stronger than that of the red fluorescence. The fluorescence which appears on the Figure is green. If R < 1, which means that green fluorescence < red fluorescence, and the fluorescence that appears on the Figure is red. B, Silencing of DRP1, but not that of ATAD3A, increased co-localization of rhPCD8 (arrows, yellow fluorescence) and the endoplasmic reticulum (ER-GFP),
suggesting that rhPCD8 was present in the ER. C, In the replicating H838 cells, some of the rhPCD8 was localized in the bulging mitochondria-associated membrane (arrows, yellow fluorescence), which was visualized by a transiently expressed human phosphotidylserine
synthase 1 (huPSS1-GFP), indicating that PCD8 was abundantly present in the
mitochondria-associated membrane of the replicating H838 cells (H838/Repl). D, In DRP1kd cells,
transiently expressed huPSS1-GFP co-localized with the rhPCD8 in the bulging MAM, suggesting that ectopically expressed PCD8 was accumulated in the enlarged MAM when formation mechanism of the transport vesicles was inhibited. Silencing of ATAD3A reduced both the size of the ER (green fluorescence) and the mitochondria. E, In the wild-type H838 cells, though some of the cytochrome c (white arrow) was present in the cytoplasm, the
majority of cytochrome c was detected in the mitochondria (upper panel). In DRP1kd cells,
however, the majority of cytochrome c was detected in the cytoplasm, and some was present in the engorged subcellular structures (central panel, white arrows). In the engorged figure,
cytochrome c seemed concentrated in a branch formation. In ATAD3Akd cells the
cytoplasmic level of cytochrome c, possibly in transport vesicles, increased markedly (lower panel, white arrowheads). F, The bulging structures were identified as
mitochondria-associated membrane as determined by huPSS1 staining. Silencing of ATAD3A however, diminished the bulging structure. G, When KDEL-conjugated GFP was used as an ER marker (upper panel), the bulged cytochrome c-positive figures did not co-localize with GFP-labeled ER (central panel), indicating that the bulged figures were mitochondria-associated
membrane, which were specialized and different from the ordinary ER. As noted previously
that ATAD3A was involved in the shipment of the transport vesicles; in ATAD3Akd cells,
level of cytochrome c increased in the cytoplasm (lower panel), confirming our previous results [20] and current hypothesis. The results are representative of at least three independent experiments, in which about 100-150 cells were analyzed.
subcellular distribution of PCD8 as determined by electron microscopy. A, Electron
microscopy analysis of the wild-type H838 cells showed (A1) the fusion of transport vesicles (white arrows), (A2) into mitochondria, which were about 50-70 nm in diameter. B, Using electron microscopy, the PCD8-specific immune-gold particles were identified in the
transport vesicle (black arrows), the transport vesicle (white arrows) that was fusing with the
mitochondrion. C, Silencing of DRP1 (DRP1kd) expression increased the number of bulging
mitochondria-associated membrane (MAM, white arrowheads) and protein aggregates at the margin of the bulging MAM. D, Using immune-gold, PCD8 (black arrows) was detected in
protein aggregates of the dilated ER/MAM (white arrowheads) in DRP1kd cells. E, Silencing
of ATAD3A (ATAD3Akd) expression increased the number of fragmented mitochondria
(black arrowheads) and transported vesicles (white arrows) around the dilated ER/MAM
(black arrow). F, In ATAD3Akd cells, the PCD8 signal was mainly present in the transport
vesicles (black arrow) in the cytoplasm or around the ER/MAM (white arrowheads). G, In
Mfn-2kd H838 cells, the presence of transport vesicles (white arrows) was more evident. M:
mitochondria; N: nucleus; H, The PCD8 signals were detected in the prospective transport
vesicles in Mfn-2kd H838 cells (black arrows), confirming our hypothesis that DRP1,
ATAD3A, and Mfn-2 were essential for the shaping of the ER and the mitochondrial, in particular in the shipment of transport vesicles containing PCD8 from the ER/MAM to the mitochondria. M: mitochondria. These results are representative of three independent experiments, in which about 10-50 cells were analyzed.
Fig. 6. A simplified sketch for the importation route of PCD8 A to the mitochondria. A, The
importation of PCD8 is from the ER, the MAM, transport vesicles to the mitochondria. B, In
DRP1kd cells, because the budding off mechanism of transport vesicles are inhibited, the
well as in the MAM, will cause the bulging of the mitochondria-associated membrane. C, In
ATAD3Akd cells, although the transport vesicles are formed around the dilated ER/MAM, the
lack of ATAD3A (ATPase for providing movement energy) will idle the movement of transport vesicles toward the mitochondria. Deficiency in supplement of the newly synthesized proteins and phospholipids will then increase the number of the fragmented mitochondria. D, Likewise, because the transport vesicles cannot fuse into mitochondria, the
presence of transport vesicles around the mitochondria is more evident in Mfn-2kd H838 cells.
The markedly increased transport vesicles about the mitochondria may be induced by a yet to be determined intracellular feedback mechanism that is essential for maintaining the
