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Targeting of the mitochondrial membrane proteins to the cell surface for functional studies
BBRC Biochemical and Biophysical Research Communications 338 (2005) 1143–1151 www.elsevier.com/locate/ybbrc
Targeting of the mitochondrial membrane proteins to the cell surface for functional studies Hossein Ardehali a,*, Tian Xue b, Peihong Dong b, Carolyn Machamer c a
b
Feinberg Cardiovascular Institute, Northwestern University, Chicago, IL 60611, USA Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA c Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Received 7 October 2005 Available online 21 October 2005
Abstract Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K+ and Na+ ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques. 2005 Elsevier Inc. All rights reserved. Keywords: Mitochondria; Protein trafficking; ATP-binding cassette proteins; Mitochondrial targeting signal; Membrane proteins
Mitochondria contain hundreds of proteins on their inner and outer membranes. These proteins carry various functions related to substrate transport, energy production, and other physiological processes of the mitochondria. Studying mitochondrial membrane proteins that are involved in substrate or ion transport is technically difficult due to limited access to the organelles using conventional molecular techniques. Several groups have used isolated mitochondria to study ion and substrate transport across the mitochondrial membranes. These studies are limited by the difficulty to patch clamp the mitochondria, potential contamination of the isolated mitochondria by proteins *
Corresponding author. Fax: +1 312 503 0137. E-mail address: [email protected] (H. Ardehali).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.070
from other organelles, and inconsistency of the obtained results. Thus, it has become crucial to devise a new strategy to study the function of mitochondrial membrane proteins that carry out substrate transport or ion channel activity. ATP-binding cassette (ABC) proteins form a large family, members of which have been isolated from many organisms [1]. These are membrane proteins that often utilize the energy from ATP hydrolysis to transport various substrates, such as amino acids, steroids, proteins, phospholipids, metals, and sugar [2], although in some cases the ATP plays a modulatory role (e.g., sulfonylurea receptor). ABC proteins are defined by the presence of the ABC unit, a 200–250 amino acid region consisting of two short Walker A and Walker B ATP-binding motifs, and a third conserved sequence called the SGGQ signature motif.
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The transmembrane domain (TMD) is usually composed of 5–10 membrane spanning domains (MSD). ABC proteins are categorized based on the number of TMDs and ABC units present. Full molecule ABC proteins contain 2 TMDs and 2 ABC units, while half-molecule proteins contain only one TMD and one ABC unit [2]. Two mitochondrial ABC (mABC1 and mABC2) proteins were recently isolated and sequenced, however, their functions have not been elucidated [3,4]. These proteins are half-molecule ABC proteins that contain a single TMD and ABC unit. The N-terminal 55 amino acids of mABC1 have been shown to function as mitochondrial targeting sequences, since they divert green fluorescent protein to the mitochondria. We recently showed that mABC1 protein is present in purified mitochondrial inner membrane fractions, and that it coimmunoprecipitates with several mitochondrial inner membrane proteins, including adenine nucleotide translocator (ANT) and ATP synthase [5]. Most mitochondrial proteins are translated in the cytoplasm and transported into the mitochondria after synthesis and release from ribosomes. This process involves translocases in the outer membrane (TOM complexes) and the inner membrane (TIM complexes) of the mitochondria [6– 8]. Most mitochondrial preproteins contain a mitochondrial targeting signal (MTS) in their N-termini, although some contain this information internally in the protein [6–10]. In addition to an MTS, inner mitochondrial membrane proteins contain a hydrophobic sequence that directs protein transport, insertion, and orientation in the mitochondrial inner membrane. These topogenic signals usually contain a hydrophobic transmembrane domain [11]. MTSs are about 20–60 amino acids in length and have abundant positive charges. They are predicted to form amphipathic a-helices in membranes and are recognized by protein import machinery [12]. Proteins containing these peptides fused to non-mitochondrial proteins are imported into mitochondria [13–15]. The role of these sequences in mitochondrial protein trafficking has also been studied using mutational analysis. It is believed that mitochondrial proteins are released from ribosomes into the cytoplasm as completed chains. A number of cytosolic proteins interact with these nascent proteins and guide them to the mitochondria [12]. Here, we decided to target two mitochondrial membrane proteins, mABC1 and mABC2 to the cell surface for functional studies. The removal of the MTS in these proteins led to distinct subcellular localizations. In case of mABC1, the truncated protein lacking MTS was detected throughout the secretory pathway, i.e., on the cell surface, in the ER and the Golgi complex. However, removal of the MTS in mABC2 caused protein aggregates inside the cell. Patch clamp of cells expressing mABC1 on their surface exhibited nonselective transport of K+ and Na+ ions, and resulted in the loss of membrane potential. Our results may provide a new approach for studying mitochondrial membrane proteins by targeting them to the cell surface, where the protein is more accessible for various molecular and cellular studies.
Materials and methods Chemicals and reagents. All of the chemicals and reagents were purchased from Sigma (St. Louis, MO) unless stated otherwise. Construction of expression vectors. Human mABC1 and mABC2 cDNA plasmids were kindly provided by Dr. Victor Ling (British Columbia Cancer Research Center, Vancouver, Canada). The plasmids were initially sequenced. Nine base pair changes in mABC1 and one in mABC2 were noted when compared with the published sequences [3,4]. These differences are also present in the human genome sequence, suggesting that the published sequences are incorrect. Oligonucleotides were synthesized in the Johns Hopkins DNA Synthesis Facility. To make the plasmid for full-length mABC1-GFP fusion protein, the cDNA of mABC1 without the stop codon was amplified by PCR with EcoRI and BamHI engineered at its 5 0 - and 3 0 -termini. A Kozak sequence was also engineered at the N-terminus of all clones to enhance their expression. The PCR fragment was cloned into TOPO Vector (Invitrogen), cut with EcoRI and BamHI, and cloned into pGFPN1 (Clontech). For the plasmid encoding the truncated mABC1-GFP fusion protein, a PCR product lacking the first 55 amino acids of mABC1 was synthesized and cloned into TOPO and pGFP as described above. For mABC2-GFP fusion protein plasmid, the mABC2 cDNA lacking a stop codon was amplified by PCR with EcoRI and BamHI at its 5 0 - and 3 0 termini. The PCR product was first cloned into TOPO vector and after cutting with EcoRI and BamHI was cloned into pGFP-N1. A PCR fragment lacking the first 101 amino acids of mABC2 was amplified and cloned into TOPO and subsequently pGFP-N1 to construct the truncated mABC2 plasmid. A plasmid containing mABC2 cDNA lacking only the first 55 amino acids was also constructed using PCR cloning technique as described above. An oligonucleotide containing 54 bp of the human serum albumin signal sequence (SASS) (5 0 -ATGAAGTGGGTAACCTTTATTTCCC TTCTTTTTCTCTTTAGCTCGGCTTATTCC-3 0 ) was synthesized and used as a template to amplify a fragment with BglII and EcoRI at its 5 0 and 3 0 -termini. The PCR product was cloned into pGFP-N1 and was used for subsequent cloning of mABC1, mABC2, and ANT. For fusion proteins of the transmembrane domain of CD8 (TCD8), the first 666 bp of CD8 containing its transmembrane domain was amplified by PCR with BglII and EcoRI engineered at its 5 0 - and 3 0 -termini. Both CD8 and SASS contained a Kozak sequence before the ATG start codon to enhance protein expression. TCD8 and SASS were subcloned 5 0 of the full-length mABC1 and mABC2 cDNAs. These plasmids contained GFP cDNA 3 0 of the mABC1 and mABC2 cDNAs. A summary of all mABC1 and mABC2 constructs used in our studies, along with their subcellular localization, is shown in Fig. 1. pTE1-Myc was a generous gift from Dr. Stephen J. GuoldÕs laboratory (Johns Hopkins Medical School). It contains peroxisomal acyl-coA thioesterase fused to an N-terminal Myc epitope in pcDNA3 plasmid (Invitrogen). Cell culture and transfection. Human embryonic kidney (HEK293) and HeLa cells were cultured in DulbeccoÕs modified medium (DMEM) with 10% fetal bovine serum. Cells were split onto glass-bottomed dishes, and allowed to grow to 80% confluency prior to transfection. Transfections were performed by Lipofectamine Plus reagent as described by the manufacturer (Invitrogen). About 1.5 lg of plasmid DNA was used for all transfections. Cells were analyzed by confocal microscopy about 18 h after transfection. Confocal imaging. To monitor the mitochondria, cells were loaded with 100 nmol/L of tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes) at 37 C for 15 min. After washing the dye with phosphate-buffered saline (PBS), cells were placed in phenol-red-free DMEM supplemented with 25 mmol/L Hepes. Cells were illuminated with 488 nm line and 568 nm line of a krypton/argon laser, and images were taken by confocal microscopy (PCM-2000; Nikon, Tokyo, Japan or UltraVIEW; Perkin-Elmer, Boston, MA).
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Fig. 1. Summary of the mABC1 and mABC2 constructs used in this study. Major subcellular location of the proteins is also listed, which is discussed in the text.
Immunofluorescence staining. HEK293 and HeLa cells were grown on glass slides to 75% confluency and transfected as described above. About 24 h after transfection, cells were washed in PBS twice and fixed at room temperature in 3% paraformaldehyde for 10 min. They were then permeabilized with 0.5% Triton X-100 or saponin for 3 min and blocked with PBS/glycine for 10 min. Cells were incubated in the presence of the diluted primary antibody overnight (see below). After washing the slides with PBS, cells were incubated with Alexa Fluor 546 anti-rabbit IgG (Molecular Probes, diluted 1:1000) or Alexa Fluor 546 anti-mouse IgG (Molecular Probes, diluted 1:1000). Cells were examined by a laser scanning confocal microscope using a 40· oil-immersion lens and 2· optical zoom. Alexa 546 was excited and visualized by a helium/neon laser (543 nm). Antibodies. Anti-rabbit calreticulin (Sigma, diluted 1:200), mouse monoclonal anti-b-COP (Sigma, diluted 1:20), and mouse monoclonal anti-c-Adaptin (Sigma, diluted 1:100) were diluted in a solution containing 0.1% Tween 20 and 5% dried milk in PBS. Protein disulfide isomerase (PDI) antibody was purchased from StressGen Biotechnologies (Canada) and lysosomal associated membrane protein (LAMP)-1 antibodies were obtained from Developmental Studies Hybridoma Bank (University of Iowa). Anti-myc polyclonal antibody was purchased from Boehringer– Mannhein. Immunoblotting. Twenty-four hours after cells were transfected with truncated mABC1, washed in PBS, and incubated for 30 min on ice in lysis buffer [68 mmol/L sucrose, 200 mmol/L mannitol, 50 mmol/L KCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L DTT, and one Complete Mini Protease Inhibitor Tablet (Roche)]. Cells were then homogenized twice with an ultrasound sonicator and centrifuged at 4 C (500g) to remove nuclei, unbroken cells, and debris. The supernatant was centrifuged at 14,000g for 15 min. The supernatant was then subjected to 1.5 h of centrifugation at 100,000g to pellet the light membranes. Lysates were then applied on NuPAGE 4–12% bis-Tris gels and transferred to nitro-
cellulose membrane (Invitrogen, USA). The membranes were probed with the primary antibody against GFP (Santa Cruz Biotechnology, USA) at 1:200 dilution. Electrophysiology. Whole-cell patch clamp was performed at room temperature sing an Axopatch 200B amplifier and pClamp8.0 software (Axon Inc.). Patch pipettes were prepared from 1.5 mm thin-walled borosilicate glass tubes using a Sutter micropipette puller P-97 and had typical resistances of 3–5 MX when filled with intracellular solution. Intracellular solutions contained (in mM): 140 mM KCl, 4 mM MgATP, 5 mM EGTA, 1 mM MgCl2, and 10 mM Hepes, pH 7.4 with KOH. Extracellular solutions contained (in mM): 138 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM Glc, 05 mM MgCl2, and 10 mM Hepes, pH adjusted to 7.4 with NaOH. For recording the membrane potential, the cells were held at I = 0 lA.
Results mABC1 and mABC2 localize to the mitochondria We first evaluated subcellular localization of full-length mABC1 and mABC2 proteins. HEK293T and HeLa cells were transfected with plasmids encoding GFP fusion proteins of full-length mABC1 and mABC2 proteins. As shown in Figs. 2A and B, both of these proteins colocalize with mitochondrial marker TMRE in HeLa cells. Similar results were obtained in HEK293 cells (data not shown). The cells overexpressing these proteins did not exhibit any other unusual features, suggesting that overexpression of these proteins is probably not deleterious to cells.
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Fig. 2. mABC1 and mABC2 proteins localize to the mitochondria. Confocal images of HeLa cells transfected with GFP fusion proteins of full-length mABC1 and mABC2 are shown. (A) Cells transfected with mABC1-GFP fusion protein were stained with a red fluorescence mitochondrial marker, TMRE, and the same field was acquired separately with a green (right panel) and Texas red filter (middle panel). Merged image of the two acquisitions is shown in the right panel. Yellow staining indicates overlap. (B) Cells transfected with mABC2-GFP fusion proteins and images of the same field were obtained using green (left panel) and red (middle panel) filters. Merged image of the two acquisitions is shown in the left panel. Similar results were obtained in HEK293 cells (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Removal of MTS in mABC1 results in protein targeting to the secretory pathway
ER, Golgi complex, and plasma membrane) if the mitochondrial targeting signal is removed.
In order to better understand the role of MTS in mitochondrial membrane protein trafficking, we made GFP fusion proteins of truncated mABC1 lacking the N-terminal MTS. Truncated mABC1 was mostly detected on the cell surface in HEK293 cells. Furthermore, HEK293 cells expressing mABC1 on the cell surface did not take up the mitochondrial marker TMRE (Fig. 3A). The reason for this phenomenon is not clear at this point, and will be discussed later. In HeLa cells, the truncated mABC1 was also detected on the cell surface, however intracellular green fluorescence was also detected (Fig. 3B). We then used immunohistochemistry to study the subcellular localization of the truncated mABC1 in HeLa cells. The green fluorescence of truncated mABC1-GFP protein colocalized with the Golgi marker, c-adaptin, and the ER marker, PDI (Figs. 3C and D). Similar results were obtained when a component of COPI-coated vesicles, b-COP, was used (data not shown). However, the truncated mABC1 did not localize with LAMP-1 (data not shown), suggesting that the protein is not present in the lysosomes. We also studied the cellular location of the truncated mABC1 protein by subcellular fractionation and Western blot analysis (Fig. 4). About 60% of the truncated mABC1 protein was detected in the membranes, while the rest was present in the cytoplasmic fraction. These results suggest that mABC1 can go through the secretory pathway (i.e.,
Removal of MTS in mABC2 results in protein aggregation inside the cell While truncated mABC1 is mostly present on the cell surface and secretory pathway organelles, mABC2 lacking the MTS formed large globules inside the cells in HEK293 and HeLa cells (Figs. 5A and B, respectively). The truncated mABC2-GFP fusion protein was rarely detected on the cell surface or in mitochondria. Western blot analysis of subcellular fractions revealed that close to 80% of the truncated mABC2 is present in the cytoplasm, with the remaining 20% associated with membranes (Fig. 4). The putative MTS for mABC2 encompasses the N-terminal 101 amino acids of this protein [3]. This is longer than the 55 amino acid segment that is thought to contain the MTS of mABC1. Thus, one may argue that the difference between subcellular localization of the truncated mABC1 and mABC2 may be due to the loss of some other element in mABC2 which may reside in its amino acids 55– 101. In order to explore this possibility, we made a GFP fusion protein of truncated mABC2 lacking only the first 55 amino acids. Cells overexpressing this truncated protein exhibited features similar to those of truncated mABC2 lacking 101 amino acids, i.e., large globules within the cells (data not shown). Thus, disruption of the MTS in mABC2 by deletion leads to its aggregation within cells.
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Fig. 3. Removal of MTS from mABC1 results in protein localization on the cell surface and in secretory pathway organelles. (A) Confocal images of HEK293 cells transfected with the truncated forms of mABC1. Cells transfected with a truncated mABC1 protein (lacking the MTS) display green fluorescence mostly on their cell surfaces (left panel). Cells were also stained with TMRE (middle panel). Merged image of the green and red acquisitions is shown in the right panel. (B) Confocal image of HeLa cells transfected with the truncated form of mABC1. Unlike HEK 293 cells, where most of the green fluorescence was noted on the cell surface, HeLa cells also displayed some green fluorescence within the cell. This did not localize with TMRE. Immunohistochemistry of the cells revealed that the green fluorescence localizes with the Golgi marker, c-adaptin (C), and the ER marker, PDI (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
The exact location of truncated mABC2 proteins lacking MTS could not be derived from confocal imaging. It is possible that the transport of these proteins is prematurely arrested in the ER. We thus used immunohistochemistry to examine whether these protein globules containing the truncated mABC2 colocalize with ER markers. As shown in Figs. 6A and B, truncated mABC2-GFP fusion protein did not colocalize with the ER markers PDI and calreticulin. We then asked whether these aggregates represent endosomes, lysosomes or peroxisomes. Cells were first stained with LAMP-1, which is a lysosomal/endosomal marker. Green fluorescence failed to colocalize with LAMP-1 red fluorescence (Fig. 6C), suggesting that these globules do not represent endosomes or lysosomes. To label peroxisomes, we transfected cells with a plasmid encoding a peroxisome protein (peroxisomal acyl-coA thi-
oesterase) fused to an N-terminal myc epitope. The cells were then probed with an antibody against the myc tag. As shown in Fig. 6C, the green fluorescence did not localize with the peroxisome marker either. Thus, truncated mABC2 protein forms aggregates within the cell, and does not localize with the mitochondria, ER, endosomes, lysosomes or peroxisomes. Influence of SASS and TCD8 on localization of mitochondrial membrane proteins We then asked how the addition of a signal sequence or the transmembrane domain of CD8 to the N-terminus of mitochondrial membrane proteins would influence their trafficking. SASS-mABC1 and TCD8-mABC1 fusion proteins were mostly detected on the cell surface and cytoplasm
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Fig. 4. Western blot analysis of subcellular fractions of cells transfected with truncated mABC1 and mABC2. About 60% of the truncated mABC1 was detected on the light membranes and the rest was in cytoplasm, while about 80% of the truncated mABC2 was in the cytoplasm.
(Figs. 7A and B). However, SASS and TCD8 fusion proteins of mABC2 were again detected as large globules inside the cell, similar to what was observed with truncated mABC2 (Figs. 7C and D). The influence of SASS on protein localization was less prominent than that of CD8. Addition of SASS and TCD8 to the N-terminus of the truncated forms of mABC1 and mABC2 also resulted in similar cellular localization as with the full-length proteins. The results of these studies are summarized in Fig. 1.
Patch clamp of truncated mABC1 (TmABC1) transfected cells We then studied HEK cells expressing mABC1 on their surface for ion transport activity using patch clamp technique. The TmABC1 transfected cells were identified under fluorescence microscopy and were patch clamped in bathing solutions containing different K+ concentrations. As shown in Fig. 8, there was passive movement of ions in cells expressing the truncated mABC1 on their surface. Membrane potential of the cells was also measured and compared to that of GFP-only transfected cells. TmABC1 transfected cells displayed current with zero reversal potential. We also performed the studies in the presence of a mitochondrial ATP-sensitive K+ channel inhibitor, 5hydroxydeconate. The addition of this chemical did not affect patch clamp results. These results suggest that mABC1 may function as a nonselective pore, although other possibilities (such as toxic effects of mABC1 overexpression on the cells) may also explain the observed results. Thus, further studies will be needed to fully elucidate the function of these proteins. Discussion Studying mitochondrial transport proteins and ion channels has proved to be a technical challenge. Here, we attempted to target two mitochondrial ABC proteins to the cell surface in order to study their functions. We showed that removal of the MTS in mABC1 localizes the protein to the cell surface or the secretory pathway, while a truncated mABC2 lacking a MTS forms cellular aggregates. Furthermore, addition of a cell membrane signal sequence or the transmembrane domain of CD8 targeted mABC1 to the cell surface, while fusion proteins of
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Fig. 5. Truncated mABC2 protein, lacking an MTS, forms aggregates inside HEK293 (A) and HeLa cells (B). The middle panels represent TMRE stained cells imaged with red filter, and the right panel is merged image of green and red acquisition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
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Fig. 6. Immunohistochemistry of HeLa cells transfected with the truncated mABC2 and stained with ER, lysosomal or peroxisome markers. (A) Represents cells stained with ER markers, PDI. Green fluorescence failed to localize with this marker. (B) Green fluorescence did not localize with lysosome/endosome marker, LAMP-1, either. (C) Cells were transfected with a plasmid containing a peroxisome protein, peroxisomal acyl-coA thioesterase fused to an N-terminal myc epitope, and probed with a myc antibody. The green fluorescence again did not localize with the peroxisome marker. Similar results were obtained in HEK293 cells (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
mABC2 were retained inside the cell in aggregates. Since the orientation of mABC1 in the mitochondria is unknown, we cannot speculate how these manipulations affect the topology of this protein when it is targeted to the secretory pathway. Cells expressing mABC1 on their surface displayed 0 mV membrane potential and passive movement of ions. The results obtained suggest that mABC1 may function as a nonselective pore, or the observed cellular changes could be through the nonspecific effects of mABC1 overexpression on the cells. To elucidate the function of this protein, we are currently performing more comprehensive functional studies on mABC1. It is intriguing that deletion of the MTS in mABC2 resulted in protein aggregation within the cell. One possibility is that the truncated mABC2 lacks the primary structure needed for adequate folding of the protein. These findings suggest that in mABC2, and possibly other mitochondrial membrane proteins, the MTS contains additional information which may assist in maintaining a viable structure for the protein. The additional information would also be disrupted upon the addition of another sequence in the N-terminus of the protein, as was observed with SASS and TCD8. Thus, in addition to its role in targeting proteins
to the mitochondria, certain amino acids within the MTS may play a crucial role in the overall structure of the protein. At this time, we do not know which amino acids within the MTS are responsible for protein structure, and which ones target the protein to the mitochondria. Here, we showed that the addition of a signal sequence or the transmembrane domain of CD8 can target mitochondrial membrane proteins to the cell surface. Thus, it is possible that the protein transport machinery recognizes the initial data in the N-terminus of the protein and prioritizes that information over anything else that resides within the protein. A similar scenario has been proposed for the cell membrane proteins where the signal recognition particle recognizes the signal sequence as it gets translated by the ribosomal complex [16]. How this is done in mitochondrial proteins is not clear yet. In experiments not shown here, we have demonstrated that the addition of TCD8 and a signal sequence can also target a protein with an internal MTS, adenine nucleotide translocator, to the cell surface. Analysis of the function of mitochondrial membrane proteins, including those with an internal MTS, has been limited to in vitro systems, since it has not been possible to direct them to the cell surface and study their
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function when they are expressed on the eukaryotic cell membranes. Our findings may open new ways to study these proteins in established cell culture systems by retargeting them to the cell surface. In summary, the present findings suggest differential subcellular localization of different mitochondrial inner membrane proteins upon the removal of their MTSs. In the case of mABC1, the removal of MTS or addition of a SS or CD8 TMD leads to cell surface or secretory pathway localization, while similar modifications of mABC2 cause aggregation of the protein inside the cell. Our findings open a new approach to study mitochondrial membrane proteins by targeting them to the cell surface, where various molecular and cellular techniques can be used to study their function. Acknowledgments
Fig. 7. Addition of SASS and TM domain of CD8 directs mABC1 to the cell surface and causes aggregation of mABC2. HEK293 cells were transfected with constructs containing SASS or TCD8 upstream from mABC1-GFP or mABC2-GFP fusion proteins and analyzed by confocal microscopy. Cells expressing SASS- and TCD8-mABC1 fusion proteins are shown in (A,B), respectively. SASS- and TCD8-mABC2 are shown in (C,D).
We are indebted to Dr. Eduardo Marba´n (Michel Mirowski, MD Professorship of Cardiology, Johns Hopkins University) for his support of this study and helpful comments. This study was supported in part by the NIH Grant R37 HL36957 to Dr. Marba´n. H.A. is supported by the Competitive Grant Awards Program for Young Investigators by GlaxoSmithKline Research and Educa-
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Fig. 8. Patch clamp of truncated mABC1 and GFP transfected HEK cells. For patch clamp studies, the transfected cells were first identified by fluorescence microscopy. (A) Patch clamp of truncated mABC1 transfected cells showed passive membrane conductance, while GFP only transfected cells (controls) did not display significant current. A representative experiment is shown in (B). These experiments were performed with K+ as the predominant cation in the solution. Addition of Na+ also resulted in similar results.
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tion Foundation for Cardiovascular Disease and the NIH K08 HL79387. References [1] I. Klein, B. Sarkadi, A. Varadi, An inventory of the human ABC proteins, Biochim. Biophys. Acta 1461 (1999) 237–262. [2] S. Childs, V. Ling, The MDR superfamily of genes and its biological implications, in: V.T. DeVita, S. Hellman, S. Rosenburg (Eds.), Important Advances in Oncology, Lippincott Company, Philadelphia, 1994. [3] F. Zhang, D.L. Hogue, L. Liu, C.L. Fisher, D. Hui, S. Childs, V. Ling, M-ABC2, a new human mitochondrial ATP-binding cassette membrane protein, FEBS Lett. 478 (2000) 89–94. [4] D.L. Hogue, L. Liu, V. Ling, Identification and characterization of a mammalian mitochondrial ATP-binding cassette membrane protein, J. Mol. Biol. 285 (1999) 379–389. [5] H. Ardehali, Z. Chen, Y. Ko, R. Mejia-Alvarez, E. Marban, Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity, Proc. Natl. Acad. Sci. USA 101 (2004) 11880–11885. [6] R. Stuart, Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex, Biochim. Biophys. Acta 1592 (2002) 79–87. [7] N. Pfanner, A. Geissler, Versatility of the mitochondrial protein import machinery, Nat. Rev. Mol. Cell Biol. 2 (2001) 339–349.
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[8] J. Rassow, N. Pfanner, The protein import machinery of the mitochondrial membranes, Traffic 1 (2000) 457–464. [9] C.M. Koehler, Protein translocation pathways of the mitochondrion, FEBS Lett. 476 (2000) 27–31. [10] M.F. Bauer, S. Hofmann, W. Neupert, M. Brunner, Protein translocation into mitochondria: the role of TIM complexes, Trends Cell Biol. 10 (2000) 25–31. [11] R.A. Stuart, W. Neupert, Topogenesis of inner membrane proteins of mitochondria, Trends Biochem. Sci. 21 (1996) 261– 267. [12] W. Neupert, Protein import into mitochondria, Annu. Rev. Biochem. 66 (1997) 863–917. [13] E.C. Hurt, B. Pesold-Hurt, K. Suda, W. Oppliger, G. Schatz, The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix, EMBO J. 4 (1985) 2061–2068. [14] E.C. Hurt, B. Pesold-Hurt, G. Schatz, The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix, FEBS Lett. 178 (1984) 306–310. [15] A.L. Horwich, F. Kalousek, I. Mellman, L.E. Rosenberg, A leader peptide is sufficient to direct mitochondrial import of a chimeric protein, EMBO J. 4 (1985) 1129–1135. [16] R.M. Stroud, P. Walter, Signal sequence recognition and protein targeting, Curr. Opin. Struct. Biol. 9 (1999) 754–759.
Targeting of the mitochondrial membrane proteins to the cell surface for functional studies Hossein Ardehali a,*, Tian Xue b, Peihong Dong b, Carolyn Machamer c a
b
Feinberg Cardiovascular Institute, Northwestern University, Chicago, IL 60611, USA Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA c Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Received 7 October 2005 Available online 21 October 2005
Abstract Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K+ and Na+ ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques. 2005 Elsevier Inc. All rights reserved. Keywords: Mitochondria; Protein trafficking; ATP-binding cassette proteins; Mitochondrial targeting signal; Membrane proteins
Mitochondria contain hundreds of proteins on their inner and outer membranes. These proteins carry various functions related to substrate transport, energy production, and other physiological processes of the mitochondria. Studying mitochondrial membrane proteins that are involved in substrate or ion transport is technically difficult due to limited access to the organelles using conventional molecular techniques. Several groups have used isolated mitochondria to study ion and substrate transport across the mitochondrial membranes. These studies are limited by the difficulty to patch clamp the mitochondria, potential contamination of the isolated mitochondria by proteins *
Corresponding author. Fax: +1 312 503 0137. E-mail address: [email protected] (H. Ardehali).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.070
from other organelles, and inconsistency of the obtained results. Thus, it has become crucial to devise a new strategy to study the function of mitochondrial membrane proteins that carry out substrate transport or ion channel activity. ATP-binding cassette (ABC) proteins form a large family, members of which have been isolated from many organisms [1]. These are membrane proteins that often utilize the energy from ATP hydrolysis to transport various substrates, such as amino acids, steroids, proteins, phospholipids, metals, and sugar [2], although in some cases the ATP plays a modulatory role (e.g., sulfonylurea receptor). ABC proteins are defined by the presence of the ABC unit, a 200–250 amino acid region consisting of two short Walker A and Walker B ATP-binding motifs, and a third conserved sequence called the SGGQ signature motif.
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The transmembrane domain (TMD) is usually composed of 5–10 membrane spanning domains (MSD). ABC proteins are categorized based on the number of TMDs and ABC units present. Full molecule ABC proteins contain 2 TMDs and 2 ABC units, while half-molecule proteins contain only one TMD and one ABC unit [2]. Two mitochondrial ABC (mABC1 and mABC2) proteins were recently isolated and sequenced, however, their functions have not been elucidated [3,4]. These proteins are half-molecule ABC proteins that contain a single TMD and ABC unit. The N-terminal 55 amino acids of mABC1 have been shown to function as mitochondrial targeting sequences, since they divert green fluorescent protein to the mitochondria. We recently showed that mABC1 protein is present in purified mitochondrial inner membrane fractions, and that it coimmunoprecipitates with several mitochondrial inner membrane proteins, including adenine nucleotide translocator (ANT) and ATP synthase [5]. Most mitochondrial proteins are translated in the cytoplasm and transported into the mitochondria after synthesis and release from ribosomes. This process involves translocases in the outer membrane (TOM complexes) and the inner membrane (TIM complexes) of the mitochondria [6– 8]. Most mitochondrial preproteins contain a mitochondrial targeting signal (MTS) in their N-termini, although some contain this information internally in the protein [6–10]. In addition to an MTS, inner mitochondrial membrane proteins contain a hydrophobic sequence that directs protein transport, insertion, and orientation in the mitochondrial inner membrane. These topogenic signals usually contain a hydrophobic transmembrane domain [11]. MTSs are about 20–60 amino acids in length and have abundant positive charges. They are predicted to form amphipathic a-helices in membranes and are recognized by protein import machinery [12]. Proteins containing these peptides fused to non-mitochondrial proteins are imported into mitochondria [13–15]. The role of these sequences in mitochondrial protein trafficking has also been studied using mutational analysis. It is believed that mitochondrial proteins are released from ribosomes into the cytoplasm as completed chains. A number of cytosolic proteins interact with these nascent proteins and guide them to the mitochondria [12]. Here, we decided to target two mitochondrial membrane proteins, mABC1 and mABC2 to the cell surface for functional studies. The removal of the MTS in these proteins led to distinct subcellular localizations. In case of mABC1, the truncated protein lacking MTS was detected throughout the secretory pathway, i.e., on the cell surface, in the ER and the Golgi complex. However, removal of the MTS in mABC2 caused protein aggregates inside the cell. Patch clamp of cells expressing mABC1 on their surface exhibited nonselective transport of K+ and Na+ ions, and resulted in the loss of membrane potential. Our results may provide a new approach for studying mitochondrial membrane proteins by targeting them to the cell surface, where the protein is more accessible for various molecular and cellular studies.
Materials and methods Chemicals and reagents. All of the chemicals and reagents were purchased from Sigma (St. Louis, MO) unless stated otherwise. Construction of expression vectors. Human mABC1 and mABC2 cDNA plasmids were kindly provided by Dr. Victor Ling (British Columbia Cancer Research Center, Vancouver, Canada). The plasmids were initially sequenced. Nine base pair changes in mABC1 and one in mABC2 were noted when compared with the published sequences [3,4]. These differences are also present in the human genome sequence, suggesting that the published sequences are incorrect. Oligonucleotides were synthesized in the Johns Hopkins DNA Synthesis Facility. To make the plasmid for full-length mABC1-GFP fusion protein, the cDNA of mABC1 without the stop codon was amplified by PCR with EcoRI and BamHI engineered at its 5 0 - and 3 0 -termini. A Kozak sequence was also engineered at the N-terminus of all clones to enhance their expression. The PCR fragment was cloned into TOPO Vector (Invitrogen), cut with EcoRI and BamHI, and cloned into pGFPN1 (Clontech). For the plasmid encoding the truncated mABC1-GFP fusion protein, a PCR product lacking the first 55 amino acids of mABC1 was synthesized and cloned into TOPO and pGFP as described above. For mABC2-GFP fusion protein plasmid, the mABC2 cDNA lacking a stop codon was amplified by PCR with EcoRI and BamHI at its 5 0 - and 3 0 termini. The PCR product was first cloned into TOPO vector and after cutting with EcoRI and BamHI was cloned into pGFP-N1. A PCR fragment lacking the first 101 amino acids of mABC2 was amplified and cloned into TOPO and subsequently pGFP-N1 to construct the truncated mABC2 plasmid. A plasmid containing mABC2 cDNA lacking only the first 55 amino acids was also constructed using PCR cloning technique as described above. An oligonucleotide containing 54 bp of the human serum albumin signal sequence (SASS) (5 0 -ATGAAGTGGGTAACCTTTATTTCCC TTCTTTTTCTCTTTAGCTCGGCTTATTCC-3 0 ) was synthesized and used as a template to amplify a fragment with BglII and EcoRI at its 5 0 and 3 0 -termini. The PCR product was cloned into pGFP-N1 and was used for subsequent cloning of mABC1, mABC2, and ANT. For fusion proteins of the transmembrane domain of CD8 (TCD8), the first 666 bp of CD8 containing its transmembrane domain was amplified by PCR with BglII and EcoRI engineered at its 5 0 - and 3 0 -termini. Both CD8 and SASS contained a Kozak sequence before the ATG start codon to enhance protein expression. TCD8 and SASS were subcloned 5 0 of the full-length mABC1 and mABC2 cDNAs. These plasmids contained GFP cDNA 3 0 of the mABC1 and mABC2 cDNAs. A summary of all mABC1 and mABC2 constructs used in our studies, along with their subcellular localization, is shown in Fig. 1. pTE1-Myc was a generous gift from Dr. Stephen J. GuoldÕs laboratory (Johns Hopkins Medical School). It contains peroxisomal acyl-coA thioesterase fused to an N-terminal Myc epitope in pcDNA3 plasmid (Invitrogen). Cell culture and transfection. Human embryonic kidney (HEK293) and HeLa cells were cultured in DulbeccoÕs modified medium (DMEM) with 10% fetal bovine serum. Cells were split onto glass-bottomed dishes, and allowed to grow to 80% confluency prior to transfection. Transfections were performed by Lipofectamine Plus reagent as described by the manufacturer (Invitrogen). About 1.5 lg of plasmid DNA was used for all transfections. Cells were analyzed by confocal microscopy about 18 h after transfection. Confocal imaging. To monitor the mitochondria, cells were loaded with 100 nmol/L of tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes) at 37 C for 15 min. After washing the dye with phosphate-buffered saline (PBS), cells were placed in phenol-red-free DMEM supplemented with 25 mmol/L Hepes. Cells were illuminated with 488 nm line and 568 nm line of a krypton/argon laser, and images were taken by confocal microscopy (PCM-2000; Nikon, Tokyo, Japan or UltraVIEW; Perkin-Elmer, Boston, MA).
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Fig. 1. Summary of the mABC1 and mABC2 constructs used in this study. Major subcellular location of the proteins is also listed, which is discussed in the text.
Immunofluorescence staining. HEK293 and HeLa cells were grown on glass slides to 75% confluency and transfected as described above. About 24 h after transfection, cells were washed in PBS twice and fixed at room temperature in 3% paraformaldehyde for 10 min. They were then permeabilized with 0.5% Triton X-100 or saponin for 3 min and blocked with PBS/glycine for 10 min. Cells were incubated in the presence of the diluted primary antibody overnight (see below). After washing the slides with PBS, cells were incubated with Alexa Fluor 546 anti-rabbit IgG (Molecular Probes, diluted 1:1000) or Alexa Fluor 546 anti-mouse IgG (Molecular Probes, diluted 1:1000). Cells were examined by a laser scanning confocal microscope using a 40· oil-immersion lens and 2· optical zoom. Alexa 546 was excited and visualized by a helium/neon laser (543 nm). Antibodies. Anti-rabbit calreticulin (Sigma, diluted 1:200), mouse monoclonal anti-b-COP (Sigma, diluted 1:20), and mouse monoclonal anti-c-Adaptin (Sigma, diluted 1:100) were diluted in a solution containing 0.1% Tween 20 and 5% dried milk in PBS. Protein disulfide isomerase (PDI) antibody was purchased from StressGen Biotechnologies (Canada) and lysosomal associated membrane protein (LAMP)-1 antibodies were obtained from Developmental Studies Hybridoma Bank (University of Iowa). Anti-myc polyclonal antibody was purchased from Boehringer– Mannhein. Immunoblotting. Twenty-four hours after cells were transfected with truncated mABC1, washed in PBS, and incubated for 30 min on ice in lysis buffer [68 mmol/L sucrose, 200 mmol/L mannitol, 50 mmol/L KCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L DTT, and one Complete Mini Protease Inhibitor Tablet (Roche)]. Cells were then homogenized twice with an ultrasound sonicator and centrifuged at 4 C (500g) to remove nuclei, unbroken cells, and debris. The supernatant was centrifuged at 14,000g for 15 min. The supernatant was then subjected to 1.5 h of centrifugation at 100,000g to pellet the light membranes. Lysates were then applied on NuPAGE 4–12% bis-Tris gels and transferred to nitro-
cellulose membrane (Invitrogen, USA). The membranes were probed with the primary antibody against GFP (Santa Cruz Biotechnology, USA) at 1:200 dilution. Electrophysiology. Whole-cell patch clamp was performed at room temperature sing an Axopatch 200B amplifier and pClamp8.0 software (Axon Inc.). Patch pipettes were prepared from 1.5 mm thin-walled borosilicate glass tubes using a Sutter micropipette puller P-97 and had typical resistances of 3–5 MX when filled with intracellular solution. Intracellular solutions contained (in mM): 140 mM KCl, 4 mM MgATP, 5 mM EGTA, 1 mM MgCl2, and 10 mM Hepes, pH 7.4 with KOH. Extracellular solutions contained (in mM): 138 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM Glc, 05 mM MgCl2, and 10 mM Hepes, pH adjusted to 7.4 with NaOH. For recording the membrane potential, the cells were held at I = 0 lA.
Results mABC1 and mABC2 localize to the mitochondria We first evaluated subcellular localization of full-length mABC1 and mABC2 proteins. HEK293T and HeLa cells were transfected with plasmids encoding GFP fusion proteins of full-length mABC1 and mABC2 proteins. As shown in Figs. 2A and B, both of these proteins colocalize with mitochondrial marker TMRE in HeLa cells. Similar results were obtained in HEK293 cells (data not shown). The cells overexpressing these proteins did not exhibit any other unusual features, suggesting that overexpression of these proteins is probably not deleterious to cells.
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Fig. 2. mABC1 and mABC2 proteins localize to the mitochondria. Confocal images of HeLa cells transfected with GFP fusion proteins of full-length mABC1 and mABC2 are shown. (A) Cells transfected with mABC1-GFP fusion protein were stained with a red fluorescence mitochondrial marker, TMRE, and the same field was acquired separately with a green (right panel) and Texas red filter (middle panel). Merged image of the two acquisitions is shown in the right panel. Yellow staining indicates overlap. (B) Cells transfected with mABC2-GFP fusion proteins and images of the same field were obtained using green (left panel) and red (middle panel) filters. Merged image of the two acquisitions is shown in the left panel. Similar results were obtained in HEK293 cells (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Removal of MTS in mABC1 results in protein targeting to the secretory pathway
ER, Golgi complex, and plasma membrane) if the mitochondrial targeting signal is removed.
In order to better understand the role of MTS in mitochondrial membrane protein trafficking, we made GFP fusion proteins of truncated mABC1 lacking the N-terminal MTS. Truncated mABC1 was mostly detected on the cell surface in HEK293 cells. Furthermore, HEK293 cells expressing mABC1 on the cell surface did not take up the mitochondrial marker TMRE (Fig. 3A). The reason for this phenomenon is not clear at this point, and will be discussed later. In HeLa cells, the truncated mABC1 was also detected on the cell surface, however intracellular green fluorescence was also detected (Fig. 3B). We then used immunohistochemistry to study the subcellular localization of the truncated mABC1 in HeLa cells. The green fluorescence of truncated mABC1-GFP protein colocalized with the Golgi marker, c-adaptin, and the ER marker, PDI (Figs. 3C and D). Similar results were obtained when a component of COPI-coated vesicles, b-COP, was used (data not shown). However, the truncated mABC1 did not localize with LAMP-1 (data not shown), suggesting that the protein is not present in the lysosomes. We also studied the cellular location of the truncated mABC1 protein by subcellular fractionation and Western blot analysis (Fig. 4). About 60% of the truncated mABC1 protein was detected in the membranes, while the rest was present in the cytoplasmic fraction. These results suggest that mABC1 can go through the secretory pathway (i.e.,
Removal of MTS in mABC2 results in protein aggregation inside the cell While truncated mABC1 is mostly present on the cell surface and secretory pathway organelles, mABC2 lacking the MTS formed large globules inside the cells in HEK293 and HeLa cells (Figs. 5A and B, respectively). The truncated mABC2-GFP fusion protein was rarely detected on the cell surface or in mitochondria. Western blot analysis of subcellular fractions revealed that close to 80% of the truncated mABC2 is present in the cytoplasm, with the remaining 20% associated with membranes (Fig. 4). The putative MTS for mABC2 encompasses the N-terminal 101 amino acids of this protein [3]. This is longer than the 55 amino acid segment that is thought to contain the MTS of mABC1. Thus, one may argue that the difference between subcellular localization of the truncated mABC1 and mABC2 may be due to the loss of some other element in mABC2 which may reside in its amino acids 55– 101. In order to explore this possibility, we made a GFP fusion protein of truncated mABC2 lacking only the first 55 amino acids. Cells overexpressing this truncated protein exhibited features similar to those of truncated mABC2 lacking 101 amino acids, i.e., large globules within the cells (data not shown). Thus, disruption of the MTS in mABC2 by deletion leads to its aggregation within cells.
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Fig. 3. Removal of MTS from mABC1 results in protein localization on the cell surface and in secretory pathway organelles. (A) Confocal images of HEK293 cells transfected with the truncated forms of mABC1. Cells transfected with a truncated mABC1 protein (lacking the MTS) display green fluorescence mostly on their cell surfaces (left panel). Cells were also stained with TMRE (middle panel). Merged image of the green and red acquisitions is shown in the right panel. (B) Confocal image of HeLa cells transfected with the truncated form of mABC1. Unlike HEK 293 cells, where most of the green fluorescence was noted on the cell surface, HeLa cells also displayed some green fluorescence within the cell. This did not localize with TMRE. Immunohistochemistry of the cells revealed that the green fluorescence localizes with the Golgi marker, c-adaptin (C), and the ER marker, PDI (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
The exact location of truncated mABC2 proteins lacking MTS could not be derived from confocal imaging. It is possible that the transport of these proteins is prematurely arrested in the ER. We thus used immunohistochemistry to examine whether these protein globules containing the truncated mABC2 colocalize with ER markers. As shown in Figs. 6A and B, truncated mABC2-GFP fusion protein did not colocalize with the ER markers PDI and calreticulin. We then asked whether these aggregates represent endosomes, lysosomes or peroxisomes. Cells were first stained with LAMP-1, which is a lysosomal/endosomal marker. Green fluorescence failed to colocalize with LAMP-1 red fluorescence (Fig. 6C), suggesting that these globules do not represent endosomes or lysosomes. To label peroxisomes, we transfected cells with a plasmid encoding a peroxisome protein (peroxisomal acyl-coA thi-
oesterase) fused to an N-terminal myc epitope. The cells were then probed with an antibody against the myc tag. As shown in Fig. 6C, the green fluorescence did not localize with the peroxisome marker either. Thus, truncated mABC2 protein forms aggregates within the cell, and does not localize with the mitochondria, ER, endosomes, lysosomes or peroxisomes. Influence of SASS and TCD8 on localization of mitochondrial membrane proteins We then asked how the addition of a signal sequence or the transmembrane domain of CD8 to the N-terminus of mitochondrial membrane proteins would influence their trafficking. SASS-mABC1 and TCD8-mABC1 fusion proteins were mostly detected on the cell surface and cytoplasm
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Fig. 4. Western blot analysis of subcellular fractions of cells transfected with truncated mABC1 and mABC2. About 60% of the truncated mABC1 was detected on the light membranes and the rest was in cytoplasm, while about 80% of the truncated mABC2 was in the cytoplasm.
(Figs. 7A and B). However, SASS and TCD8 fusion proteins of mABC2 were again detected as large globules inside the cell, similar to what was observed with truncated mABC2 (Figs. 7C and D). The influence of SASS on protein localization was less prominent than that of CD8. Addition of SASS and TCD8 to the N-terminus of the truncated forms of mABC1 and mABC2 also resulted in similar cellular localization as with the full-length proteins. The results of these studies are summarized in Fig. 1.
Patch clamp of truncated mABC1 (TmABC1) transfected cells We then studied HEK cells expressing mABC1 on their surface for ion transport activity using patch clamp technique. The TmABC1 transfected cells were identified under fluorescence microscopy and were patch clamped in bathing solutions containing different K+ concentrations. As shown in Fig. 8, there was passive movement of ions in cells expressing the truncated mABC1 on their surface. Membrane potential of the cells was also measured and compared to that of GFP-only transfected cells. TmABC1 transfected cells displayed current with zero reversal potential. We also performed the studies in the presence of a mitochondrial ATP-sensitive K+ channel inhibitor, 5hydroxydeconate. The addition of this chemical did not affect patch clamp results. These results suggest that mABC1 may function as a nonselective pore, although other possibilities (such as toxic effects of mABC1 overexpression on the cells) may also explain the observed results. Thus, further studies will be needed to fully elucidate the function of these proteins. Discussion Studying mitochondrial transport proteins and ion channels has proved to be a technical challenge. Here, we attempted to target two mitochondrial ABC proteins to the cell surface in order to study their functions. We showed that removal of the MTS in mABC1 localizes the protein to the cell surface or the secretory pathway, while a truncated mABC2 lacking a MTS forms cellular aggregates. Furthermore, addition of a cell membrane signal sequence or the transmembrane domain of CD8 targeted mABC1 to the cell surface, while fusion proteins of
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Fig. 5. Truncated mABC2 protein, lacking an MTS, forms aggregates inside HEK293 (A) and HeLa cells (B). The middle panels represent TMRE stained cells imaged with red filter, and the right panel is merged image of green and red acquisition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
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Fig. 6. Immunohistochemistry of HeLa cells transfected with the truncated mABC2 and stained with ER, lysosomal or peroxisome markers. (A) Represents cells stained with ER markers, PDI. Green fluorescence failed to localize with this marker. (B) Green fluorescence did not localize with lysosome/endosome marker, LAMP-1, either. (C) Cells were transfected with a plasmid containing a peroxisome protein, peroxisomal acyl-coA thioesterase fused to an N-terminal myc epitope, and probed with a myc antibody. The green fluorescence again did not localize with the peroxisome marker. Similar results were obtained in HEK293 cells (data not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
mABC2 were retained inside the cell in aggregates. Since the orientation of mABC1 in the mitochondria is unknown, we cannot speculate how these manipulations affect the topology of this protein when it is targeted to the secretory pathway. Cells expressing mABC1 on their surface displayed 0 mV membrane potential and passive movement of ions. The results obtained suggest that mABC1 may function as a nonselective pore, or the observed cellular changes could be through the nonspecific effects of mABC1 overexpression on the cells. To elucidate the function of this protein, we are currently performing more comprehensive functional studies on mABC1. It is intriguing that deletion of the MTS in mABC2 resulted in protein aggregation within the cell. One possibility is that the truncated mABC2 lacks the primary structure needed for adequate folding of the protein. These findings suggest that in mABC2, and possibly other mitochondrial membrane proteins, the MTS contains additional information which may assist in maintaining a viable structure for the protein. The additional information would also be disrupted upon the addition of another sequence in the N-terminus of the protein, as was observed with SASS and TCD8. Thus, in addition to its role in targeting proteins
to the mitochondria, certain amino acids within the MTS may play a crucial role in the overall structure of the protein. At this time, we do not know which amino acids within the MTS are responsible for protein structure, and which ones target the protein to the mitochondria. Here, we showed that the addition of a signal sequence or the transmembrane domain of CD8 can target mitochondrial membrane proteins to the cell surface. Thus, it is possible that the protein transport machinery recognizes the initial data in the N-terminus of the protein and prioritizes that information over anything else that resides within the protein. A similar scenario has been proposed for the cell membrane proteins where the signal recognition particle recognizes the signal sequence as it gets translated by the ribosomal complex [16]. How this is done in mitochondrial proteins is not clear yet. In experiments not shown here, we have demonstrated that the addition of TCD8 and a signal sequence can also target a protein with an internal MTS, adenine nucleotide translocator, to the cell surface. Analysis of the function of mitochondrial membrane proteins, including those with an internal MTS, has been limited to in vitro systems, since it has not been possible to direct them to the cell surface and study their
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function when they are expressed on the eukaryotic cell membranes. Our findings may open new ways to study these proteins in established cell culture systems by retargeting them to the cell surface. In summary, the present findings suggest differential subcellular localization of different mitochondrial inner membrane proteins upon the removal of their MTSs. In the case of mABC1, the removal of MTS or addition of a SS or CD8 TMD leads to cell surface or secretory pathway localization, while similar modifications of mABC2 cause aggregation of the protein inside the cell. Our findings open a new approach to study mitochondrial membrane proteins by targeting them to the cell surface, where various molecular and cellular techniques can be used to study their function. Acknowledgments
Fig. 7. Addition of SASS and TM domain of CD8 directs mABC1 to the cell surface and causes aggregation of mABC2. HEK293 cells were transfected with constructs containing SASS or TCD8 upstream from mABC1-GFP or mABC2-GFP fusion proteins and analyzed by confocal microscopy. Cells expressing SASS- and TCD8-mABC1 fusion proteins are shown in (A,B), respectively. SASS- and TCD8-mABC2 are shown in (C,D).
We are indebted to Dr. Eduardo Marba´n (Michel Mirowski, MD Professorship of Cardiology, Johns Hopkins University) for his support of this study and helpful comments. This study was supported in part by the NIH Grant R37 HL36957 to Dr. Marba´n. H.A. is supported by the Competitive Grant Awards Program for Young Investigators by GlaxoSmithKline Research and Educa-
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Fig. 8. Patch clamp of truncated mABC1 and GFP transfected HEK cells. For patch clamp studies, the transfected cells were first identified by fluorescence microscopy. (A) Patch clamp of truncated mABC1 transfected cells showed passive membrane conductance, while GFP only transfected cells (controls) did not display significant current. A representative experiment is shown in (B). These experiments were performed with K+ as the predominant cation in the solution. Addition of Na+ also resulted in similar results.
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tion Foundation for Cardiovascular Disease and the NIH K08 HL79387. References [1] I. Klein, B. Sarkadi, A. Varadi, An inventory of the human ABC proteins, Biochim. Biophys. Acta 1461 (1999) 237–262. [2] S. Childs, V. Ling, The MDR superfamily of genes and its biological implications, in: V.T. DeVita, S. Hellman, S. Rosenburg (Eds.), Important Advances in Oncology, Lippincott Company, Philadelphia, 1994. [3] F. Zhang, D.L. Hogue, L. Liu, C.L. Fisher, D. Hui, S. Childs, V. Ling, M-ABC2, a new human mitochondrial ATP-binding cassette membrane protein, FEBS Lett. 478 (2000) 89–94. [4] D.L. Hogue, L. Liu, V. Ling, Identification and characterization of a mammalian mitochondrial ATP-binding cassette membrane protein, J. Mol. Biol. 285 (1999) 379–389. [5] H. Ardehali, Z. Chen, Y. Ko, R. Mejia-Alvarez, E. Marban, Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity, Proc. Natl. Acad. Sci. USA 101 (2004) 11880–11885. [6] R. Stuart, Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex, Biochim. Biophys. Acta 1592 (2002) 79–87. [7] N. Pfanner, A. Geissler, Versatility of the mitochondrial protein import machinery, Nat. Rev. Mol. Cell Biol. 2 (2001) 339–349.
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[8] J. Rassow, N. Pfanner, The protein import machinery of the mitochondrial membranes, Traffic 1 (2000) 457–464. [9] C.M. Koehler, Protein translocation pathways of the mitochondrion, FEBS Lett. 476 (2000) 27–31. [10] M.F. Bauer, S. Hofmann, W. Neupert, M. Brunner, Protein translocation into mitochondria: the role of TIM complexes, Trends Cell Biol. 10 (2000) 25–31. [11] R.A. Stuart, W. Neupert, Topogenesis of inner membrane proteins of mitochondria, Trends Biochem. Sci. 21 (1996) 261– 267. [12] W. Neupert, Protein import into mitochondria, Annu. Rev. Biochem. 66 (1997) 863–917. [13] E.C. Hurt, B. Pesold-Hurt, K. Suda, W. Oppliger, G. Schatz, The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix, EMBO J. 4 (1985) 2061–2068. [14] E.C. Hurt, B. Pesold-Hurt, G. Schatz, The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix, FEBS Lett. 178 (1984) 306–310. [15] A.L. Horwich, F. Kalousek, I. Mellman, L.E. Rosenberg, A leader peptide is sufficient to direct mitochondrial import of a chimeric protein, EMBO J. 4 (1985) 1129–1135. [16] R.M. Stroud, P. Walter, Signal sequence recognition and protein targeting, Curr. Opin. Struct. Biol. 9 (1999) 754–759.