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. 2023 Oct 9;62(21):3159–3165. doi: 10.1021/acs.biochem.3c00417

Cardiolipin Regulates the Activity of the Mitochondrial ABC Transporter ABCB10

Tianqi Zhang 1, Jixing Lyu 1, Yun Zhu 1, Arthur Laganowsky 1,*
PMCID: PMC10634319  PMID: 37807693

Abstract

graphic file with name bi3c00417_0005.jpg

The ATP-binding cassette (ABC) transporter ABCB10 resides in the inner membrane of mitochondria and is implicated in erythropoiesis. Mitochondria from different cell types share some specific characteristics, one of which is the high abundance of cardiolipin. Although previous studies have provided insight into ABCB10, the affinity and selectivity of this transporter toward lipids, particularly those found in the mitochondria, remain poorly understood. Here, native mass spectrometry is used to directly monitor the binding events of lipids to human ABCB10. The results reveal that ABCB10 binds avidly to cardiolipin with an affinity significantly higher than that of other phospholipids. The first three binding events of cardiolipin display positive cooperativity, which is suggestive of specific cardiolipin-binding sites on ABCB10. Phosphatidic acid is the second-best binder of the lipids investigated. The bulk lipids, phosphatidylcholine and phosphatidylethanolamine, display the weakest binding affinity for ABCB10. Other lipids bind ABCB10 with a similar affinity. Functional assays show that cardiolipin regulates the ATPase activity of ABCB10 in a dose-dependent fashion. ATPase activity of ABCB10 was also impacted in the presence of other lipids but to a lesser extent than cardiolipin. Taken together, ABCB10 has a high binding affinity for cardiolipin, and this lipid also regulates the ATPase activity of the transporter.

Introduction

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins that play essential roles in transporting a variety of molecules across the biological membrane.1 These transporters are implicated in multidrug resistance in cancer cells, and their dysfunction is associated with human diseases. ABC transporters share a similar topology with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) often containing 12 transmembrane helices. The NBDs bind and hydrolyze ATP to provide energy for the transport cycle, while the TMDs facilitate the translocation of the substrate.2 In general, ABC exporters share a common mechanism of substrate transport whereby ATP hydrolysis powers the transition from an inward-facing to an outward-facing conformation facilitating the transport of the substrate across the bilayer.3

Human ABCB10 is an ABC exporter that is found in the inner membrane of mitochondria.4,5 Mitochondria participate in several essential and specialized functions during erythroid differentiation to produce red blood cells, including energy metabolism, iron metabolism, and heme biosynthesis.6 The lack of ABCB10 function causes a profound decrease in the erythrocyte precursor cell population by triggering apoptosis. ABCB10 deletion causes mouse embryonic lethality at day 12.5 and anemia at day 10.5, before definitive erythropoiesis starts in the liver.7 The NBDs of ABCB10 are located in the matrix of the mitochondria, and the transporter facilitates the export of a substrate from the mitochondrial matrix to the intermembrane space in an ATP-dependent manner. Recent reports suggest it may transport the heme analogue zinc-mesoporphyrin rather than other heme-related molecules previously hypothesized to be transported by ABCB10, including the heme precursor aminolevulinic acid, protoporphyrin IX, and hemin.8 More recently, ABCB10 has been shown to export biliverdin from the mitochondria to the cytosol, which is then reduced in the cytosol by biliverdin reductase to form bilirubin.9

ABCB10 adopts a fold similar to other ABC transporters consisting of a short N-terminal α helix followed by six transmembrane helices. The two monomers are interconnected by a domain swap and a linker connects the TMD to the C-terminal NBD. The crystal structure of apo-ABCB10 has been determined in an open, inward-facing conformation that is similar to other exporters.10 However, ABCB10 in complex with nonhydrolyzable ATP analogues adopts an open-inward conformation that is in contrast to other transporter structures populating an open, outward-facing conformation.10

In biological membranes, lipids are not randomly distributed but rather composed of lipids specific to the organelle and plasma membrane that contribute to their shape, structure, and function.11 Each subcellular compartment of eukaryotes also has a distinct set of proteins. In mitochondria, cardiolipin (CDL), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) represent the most abundant phospholipids. CDL is a major lipid component of the inner mitochondrial membrane (IMM), whereas it remains controversial whether cardiolipin is found in the outer mitochondrial membrane (OMM).12,13 Phosphatidylinositol (PI) is present at a significantly higher percentage in the OMM than in the IMM. Phosphatidic acid (PA) is found at low levels in mitochondria. Furthermore, the distribution of lipids in the OMM and IMM is asymmetric. For example, the majority of CDL and PI are exposed to the matrix side.14,15 CDL has also been reported to stabilize the respiratory chain supercomplex,16 interact with the ADP/ATP carrier (AAC) of the IMM,17 and to be required to maintain mitochondrial and cellular iron homeostasis.18 However, the importance of mitochondrial lipids in modulating the structure and function of ABCB10 remains poorly understood.

The application of native mass spectrometry (MS) to probe membrane protein–lipid interactions provides tremendous insight into the selectivity of lipid binding.19,20 The power of the technique lies in the ability to not only preserve noncovalent interactions in the mass spectrometer but also resolve individual ligand-binding events to protein complexes.21−23 For example, coupled with an apparatus to control the temperature, thermodynamic parameters for protein–lipid interactions can be determined using native MS.24 Recently, this technique has also been employed to uncover the selectivity of phosphatidylinositide binding to a mammalian potassium channel, GIRK2.25 To date, native MS has revealed that specific protein–lipid interactions can stabilize protein complexes22,26 and allosterically modulate other interactions with proteins,27 lipids,28 and drugs.20,29 Here, we optimize the purification of human ABCB10 for native MS studies. We then characterize the interactions between ABCB10 and phospholipids using native MS, and we characterize the role of lipids in modulating ABCB10 ATPase activity.

Methods

Protein Expression and Purification

The human ABCB10 (UniProt Q9NRK6, residues 152-738) gene was amplified by polymerase chain reaction (PCR) using Q5 high-fidelity DNA polymerase (New England Biolabs, NEB) and cloned into a modified pACEBac1 vector (Geneva Biotech) using a NEBuilder HiFi DNA Assembly (NEB). The resulting plasmid expresses ABCB10 with a C-terminal Strep-tag II tag and was confirmed by DNA sequencing. The expression plasmid was transformed into E. coli DH10EmBacY following the manufacturer’s protocol (Geneva Biotech). Blue-white screening was used to identify recombinant bacterial clones. A single, white colony was grown overnight and used for bacmid preparation using the HiPure Plasmid Midiprep Kit (Invitrogen). The purified baculoviral DNA (30 μg) was incubated with PEI Max transfection reagent (Polysciences, 60 μL, 1 mg/mL)30 and 2 mL of phosphate-buffered saline (PBS) for 20 min. The mixture was added to Spodoptera frugiperda (Sf9) cells (30 mL, 0.8 × 106 cell/mL) grown in suspension and incubated at 27 °C with shaking for 7 days. The baculovirus was amplified in Sf9, and the supernatant was collected and stored at 4 °C after centrifugation (4000g, 10 min). Trichoplusia ni (Tni) cells were used for protein expression for 72 h and then harvested by centrifugation (4000g, 10 min). Cell pellets were stored at −20 °C or used directly.

Cell pellets were resuspended in lysis buffer (30 mM Tris, 300 mM NaCl, pH 7.4 at room temperature) and lysed with four passages through a Microfluidics M-110P Microfluidizer at 25,000 psi. The lysate was centrifuged at 20,000g for 25 min at 4 °C to remove insoluble material, and the supernatant was centrifuged at 100,000g for 2 h at 4 °C to pellet membranes. The pelleted membranes were resuspended in membrane resuspension buffer (30 mM Tris, 150 mM NaCl, and 10% glycerol, pH 7.4 at room temperature) and homogenized using a glass tissue homogenizer (Wheaton). Membrane proteins were extracted with the addition of 1% (w/v) n-dodecyl β-maltoside (DDM, Glycon Biochemicals) and incubated for 2 h at 4 °C with gentle agitation. The extraction was clarified by centrifugation (40,000g, 20 min) and filtered using a 0.45 μm syringe filter. The sample was then loaded onto a drip column packed with 500 μL of Strep-Tactin Sepharose (IBA Lifesciences) equilibrated with buffer A (50 mM Tris, 150 mM NaCl, 10% glycerol, pH 7.4 at room temperature and 0.025% DDM). The column was then washed with 4 column volumes (CV) of buffer A, 8 CV of buffer B (50 mM Tris, 150 mM NaCl, 10% glycerol, pH 7.4 at room temperature and 5.6 mM DHPC [1,2-diheptanoyl-sn-glycero-3-phosphocholine]), and 10 CV of buffer C (50 mM Tris, 150 mM NaCl, 10% glycerol, pH 7.4 at room temperature and 0.065% C10E5 [pentaethylene glycol monodecyl ether]). The protein was eluted with buffer D (buffer C supplemented with 3 mM desthiobiotin). The eluted protein was injected onto a HiTrap 5 mL Desalting Column (Cytiva) equilibrated with buffer C. Peak fractions containing the desalted membrane protein were collected.

Sample Preparation for MS Analysis

Following established methods,21 the purified protein was buffer exchanged into an aqueous ammonium acetate solution (200 mM ammonium acetate, 0.065% C10E5) using a Micro Bio-Spin 6 Column (Bio-Rad Laboratories) for native MS studies. The concentration of the protein was determined using a DC Protein Assay Kit (Bio-Rad) with bovine serum albumin as the protein standard, and this value was also used to determine an extinction coefficient at 280 nm of 0.25 μM–1 cm–1.

Preparation and Titration of Lipids

Lipids were purchased from Avanti Polar Lipids and prepared as previously described.24 In brief, dried lipid films were resuspended in deionized water to a desired concentration (∼5 mM). Phospholipid concentration was determined by phosphorus analysis.31 For each lipid, serial dilutions in MS buffer (200 mM ammonium acetate, 0.065% C10E5) were prepared to a concentration twice the desired concentration. For lipid titrations, the protein solution and lipid solutions were mixed at a 1:1 volume ratio and then loaded into a gold-coated borosilicate glass capillary prepared in-house. Exactive Plus EMR Orbitrap Mass Spectrometer (Thermo Scientific) was tuned as follows: spray voltage 1.70 kV, capillary temperature 200 °C, collision-induced dissociation (CID) 60 eV, collision energy (CE) 100, trapping gas pressure 6, source DC offset 25 V, injection flatapole DC 8 V, inter flatapole lens 7, bent flatapole DC 6 V, transfer multipole DC 2, C-trap entrance lens 2. Mass spectra were acquired with a resolution of 17,500, microscans of 1, and averaging of 100. Native MS data files were deconvoluted using UniDec software.32

ATPase Activity Assay

The ATPase activity of ABCB10 was determined at 37 °C following a modified version of the malachite green assay.33 Purified protein was added to a final concentration of 0.3 μM with 5 mM magnesium chloride and varied concentrations of ATP ranging from 0 to 2000 μM. For analysis of protein with lipids, lipids were added to a final concentration of 20 μM. ATP hydrolysis was stopped with the addition of MG-AM reagent (3:1 mixture of 0.045% [w/v] malachite green and 4.2% [w/v] ammonium molybdate in 4 N HCl, filtered, 0.04% [v/v] Triton X-100 added), and 34% (w/v) sodium citrate. The volume ratio of the reaction, MG-AM reagent, and sodium citrate was 2:8:1. The quenched reaction was incubated for 30 min at room temperature to allow for color development, and the absorbance at 650 nm was measured on a CLARIOstar microplate reader (BMG Labtech).

Results

Optimization of ABCB10 for Native MS Studies

With the goal of resolving individual lipid-binding events to ABCB10 using native MS, we set out to express and purify the transporter from insect cells. The native mass spectrum of ABCB10 following methods established for structural studies10 in dodecylmaltoside (DDM) detergent revealed significant sample heterogeneity (Figure 1A). High-energy instrument settings were necessary to obtain an interpretable mass spectrum on a Waters Synapt G1 instrument (Figure 1B). The dominant mass spectral peaks centered around 5000 m/z correspond to a dimer with significant adducts bound, ranging in mass from 850 to 2190 Da, even under this high-energy regime. Another set of mass spectral peaks centered around 3500 m/z corresponds to a monomer, which probably is from gas-phase dissociation of the dimer as a result of the high collisional energy settings. For both stoichiometries, the underlying broad hump indicates that the transporter is bound to heterogeneous adducts, which we speculate is the copurified lipid. It is difficult to preserve a native-like structure using DDM, a noncharge-reducing detergent, as evident by the dissociation of the complex. Similar observations have been made for other membrane proteins.22,25,34 Therefore, we detergent-exchanged ABCB10 from DDM into the charge-reducing detergent pentaethylene glycol monodecyl ether (C10E5). The mass spectrum of this sample contained a signal predominantly for dimeric ABCB10 (Figure 1C). In addition, the dimer retained bound molecules with masses of 680 and 1360 Da, consistent with the mass of phospholipids. In order to remove the copurified contaminants, immobilized ABCB10 was washed with 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), a short-chain phospholipid with detergent-like properties that has been effective for cleaning up mammalian potassium channels.25,35 The mass spectrum of ABCB10 after DHPC treatment obtained on an Orbitrap mass spectrometer36 produced a well-resolved mass spectrum with a signal only for dimeric ABCB10 (Figure 1D). In a previous study,37 we found the measured mass (64,921.97 ± 0.1 Da) of the denatured ABCB10 subunit corresponds to the theoretical mass (64,922.2 Da) of the subunit with the initiating methionine removed followed by N-terminal acetylation, a post-translational modification.38 The measured mass (129,848 ± 1 Da) of the intact dimeric complex agrees with the theoretical mass for the modified subunit (129,844 Da). Other low abundance signals corresponding to 304, 622, and 1004 Da adducts are bound to ABCB10. The optimized samples of ABCB10 enable the opportunity to interrogate the binding of small molecules, such as nucleotides and lipids, to the transporter.

Figure 1.

Figure 1

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Optimization of human ABCB10 for native MS studies. (A) Native mass spectrum of ABCB10 purified in DDM and recorded under low energy settings. (B) Native mass spectrum of ABCB10 purified in DDM and recorded under non-native, high collisional activation settings. Signals for both the monomer and dimer are present. The broad hump observed for both low and high-energy instrument settings indicates the sample is bound to heterogeneous adducts. (C) Mass spectrum of ABCB10 after detergent exchange into the C10E5 detergent. Although the mass spectral peaks are nearly baseline-resolved, the transporter is bound to several adducts with masses consistent with those of lipids. (D) The native mass spectrum of ABCB10 in C10E5 after DHPC treatment acquired on an Exactive Plus EMR Orbitrap Mass Spectrometer is significantly improved with the majority of the signal corresponding to dimeric ABCB10.

Determination of Equilibrium Dissociation Constants for ABCB10–Lipid Interactions

To better understand ABCB10–lipid interactions, we titrated the optimized samples of ABCB10 solubilized in the C10E5 detergent with different phospholipids. We selected cardiolipin (TOCDL, 1,1′,2,2′-tetraoleoyl-cardiolipin) or phospholipids harboring 1-palmitoyl-2-oleoyl (PO, 16:0–18:1) tails but with different head groups: PA, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI) for our studies. Notably, these lipids are found in the IMM of mitochondria, where ABCB10 resides. The first lipid we titrated was POPI and at the highest concentration (15 μM) up to seven binding events to ABCB10 are observed (Figure 2A). The mass spectra in this titration series are then deconvoluted using UniDec software32 prior to computing the mole fraction of apo and lipid-bound species (Figure 2B). In a similar fashion to our previous studies,24,39 a sequential lipid-binding model was fitted to the mole fraction data to determine the equilibrium dissociation constant (KdN) for the Nth lipid-binding event to ABCB10 (Figure 2B,C). In an analogous fashion, the Kd values were determined for TOCDL, POPA, POPC, POPE, POPG, and POPS (Figures S1,S2 and Table S1).

Figure 2.

Figure 2

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Determination of equilibrium dissociation constants (Kd) for individual lipid-binding events to ABCB10. (A) Representative native mass spectra of ABCB10 titrated with POPI. The final concentration of POPI is shown. (B) Plot of mole fraction data for ABCB10(POPI)0–5 determined from the titration series (dots) and resulting fit from a sequential lipid-binding model (solid lines). (C) KdN values for the Nth lipid binding to ABCB10. Reported are the mean and standard deviation (n = 3).

The binding affinities for ABCB10-lipid interactions provide a means to define the selectivity of the transporter. The bulk lipids, POPC and POPE, displayed the lowest affinity toward ABCB10 with POPC having the highest Kd values (Kd1 of 29.3 μM). ABCB10 binding of the first and, more generally, additional lipids were largely similar for POPG, POPS, and POPI. ABCB10 binds more avidly to TOCDL (2.52 ± 0.12 μM) compared to POPA with a Kd1 value of 3.28 ± 0.08 μM (student’s t-test, p < 0.05). The binding of additional POPA molecules followed trends similar to those of the other lipids with a gradual increase in Kd values for each subsequent binding event. In contrast, there is a shallow increase in binding the first to third TOCDL to ABCB10 that follows with an upward jump in value for the fourth molecule binding the transporter. This result indicates strong positive cooperativity in the first few binding events and suggests ABCB10 has specific binding sites for TOCDL.

Mutation of a CDL Site Observed in an ABCB10 Structure

In the crystal structures of ABCB10, lipid and detergent molecules are bound to the outer surface of the transmembrane helices. In the structures, a CDL facilitates crystal packing by forming a bridge between ABCB10 molecules.10 This CDL is located on the protein exterior, intermembrane-facing leaflet of the IMM. We introduced the K416A into ABCB10, a residue involved in binding CDL (Figure S3A). The level of expression of the mutant protein was significantly reduced. Furthermore, the mass spectrum showed a broad charge state distribution along with a mixture of monomeric and dimeric species (Figure S3B). These results hindered the analysis of the binding of CDL to the transporter using native MS.

Impact of Phospholipids on the ATPase Activity of ABCB10

To better understand the connection between the binding affinity of ABCB10-lipid interactions and function, we carried out activity assays of ABCB10 in the presence of ATP and different lipids. The ATPase activity of ABCB10 in the absence of lipids but in the presence of different concentrations of ATP was determined by measuring the inorganic phosphate generated upon the enzymatic hydrolysis of ATP to ADP (Figure 3A). The Michaelis–Menten equation was fitted to the initial rates to determine the Michaelis constant (KM), catalytic rate constant (kcat), and catalytic efficiency (kcat/KM) (Figure 3B–E). The same procedure was conducted for ABCB10 in the presence of 20 μM of either TOCDL, POPA, POPC, POPE, POPG, POPS, or POPI. Some of the lipids, for example, POPC, had no impact on ABCB10 ATPase activity. Interestingly, ABCB10 in the presence of POPA or TOCDL resulted in a statistically significant decrease in kcat, which was more pronounced for TOCDL. More specifically, TOCDL reduced the catalytic rate of ABCB10 by more than 3-fold compared to apo ABCB10, leading to a reduction in the catalytic efficiency of ABCB10.

Figure 3.

Figure 3

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Phospholipids selectively modulate ABCB10 ATPase activity. (A) Initial rates for apo ABCB10 ATPase activity with different concentrations of ATP obtained using an inorganic phosphate assay. (B) Michaelis–Menten plots of ABCB10 in the absence and presence of different lipids. Protein and lipid concentration was 0.3 and 20 μM, respectively. (C–E) Bar graphs of Michaelis–Menten parameters for catalytic rate (kcat), Michaelis constant (KM), and catalytic efficiency (kcat/KM). (F) Dose-dependent inhibition of ABCB10 ATPase activity by TOCDL. Reported are the mean and standard deviation (n = 3). For comparison, the student’s t-test (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001) was performed between apo ABCB10 and the asterisked lipid.

We next conducted additional experiments to examine the dependence of ABCB10 activity on the concentration of lipids. The ATPase activity of ABCB10 was inhibited upon the addition of TOCDL with IC50 at ∼10 μM and inhibition was reduced to 0.09 at the highest concentration of TOCDL (50 μM) (Figure 3F). The addition of a natural cardiolipin extract also inhibited the ATPase activity of ABCB10 to the same extent as TOCDL, indicating that the other forms of CDL can also inhibit the transporter (Figure 4). As the binding affinity for the PO-type lipids was much weaker than that for TOCDL, we performed ABCB10 activity assays at a higher concentration of lipids (Figure 4). With the exception of POPC and POPE, ATPase activity was reduced by approximately 2-fold in the presence of POPG, POPS, and POPI. However, the activity of the ABCB10 was not reduced to levels comparable to TOCDL. In summary, ABCB10 ATPase activity is inhibited by CDL, the lipid that binds avidly to the transporter, in a dose-dependent fashion.

Figure 4.

Figure 4

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ATPase activity of ABCB10 in the presence of different lipid concentrations. ABCB10 ATPase activity over an 8 min time interval in the absence and presence of lipids using an inorganic phosphate assay. The protein concentration was 0.3 μM, and different lipid concentrations are denoted by color. Reported are the mean and standard deviation (n = 3). For comparison, the student’s t-test (*p ≤ 0.05, **p ≤ 0.01) was performed between ABCB10 in the presence of TOCDL and the asterisked lipids.

Discussion

Biophysical characterization of individual lipid-binding events for ABCB10 has illuminated its selectivity toward lipids. The results reveal ABCB10 binds avidly to TOCDL with the first three binding events exhibiting Kd values ranging from 2.52 to 6.37 μM. The Kd3 values are smaller than half of the smallest Kd3 (13.6 μM) among all other lipids studied. These results provide compelling evidence that there are specific cardiolipin-binding sites on ABCB10. Although POPA also has relatively small Kds, the abundance of this lipid in the mitochondria is at low levels compared to other lipids in mitochondria.15 POPG, POPS, and POPI bind in a similar fashion to ABCB10. In contrast, bulk lipids POPC and POPE displayed the weakest affinity for ABCB10. Interestingly, the first binding event of POPC (Kd1 = 29.3 μM) is higher than Kds for other lipids, including those representing multiple lipid binding events to ABCB10. In short, native MS provides the ability to better understand ABCB10–lipid interactions.

The binding affinity measurements in combination with functional assays provide insight into how lipids regulate ABCB10. The results of the functional assay draw a direct connection to the lipid binding affinity. First, the catalytic efficiency of ABCB10 is inhibited by TOCDL, a lipid that has the highest binding affinity in a dose-dependent fashion. Here we use pure ABCB10 samples that are free of copurified contaminants. We have recently shown for a mammalian potassium channel that copurified contaminants can abolish specific lipid binding,40 highlighting the utility of native MS to guide protein purification. Mitochondria are not only enriched in CDL ranging from 10 to 15% but CDL is asymmetrically distributed in the IMM with a higher percentage on the inner leaflet facing the mitochondrial matrix.15 Interestingly, in crystal structures of ABCB10,10 a CDL can be resolved at a site that faces the intermembrane space. This leaflet of the IMM is not as enriched in CDL as the other leaflet facing the matrix.15 Further studies are warranted to better understand how CDL regulates ABCB10. Beyond TOCDL, lipids that bind weakly, namely, POPC and POPE, do not impact ABCB10 ATPase activity. POPG, POPS, and POPI with a similar binding affinity to ABCB10 also result in a reduction of ATPase activity at higher lipid concentrations. In conclusion, the results taken together demonstrate that lipids that bind avidly to ABCB10 also impact function, and identifying the specific cardiolipin-binding sites warrants further investigation.

Acknowledgments

We thank Drs. Xin Yan, Christian Hilty, and Pingwei Li for the useful discussion. This work was supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) (R01GM121751, R01GM139876, R01GM138863, and RM1GM145416) and instrumentation support (P41GM128577).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.3c00417.

  • Representative native mass spectra for ABCB10 with lipids, plots of mole fraction data for ABCB10 with different lipids, mass spectrum for ABCB10K416A, and table of the Kds (PDF)

Accession Codes

ABCB10: Q9NRK6

Author Contributions

T.Z. and A.L. designed the research. T.Z. expressed and purified the protein. T.Z. performed the experiments. T.Z. and A.L. analyzed the data. T.Z. and A.L. wrote the manuscript with input from the other authors.

The authors declare no competing financial interest.

Supplementary Material

bi3c00417_si_001.pdf (749.7KB, pdf)

References

  1. Higgins C. F. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 1992, 8, 67–113. 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  2. Locher K. P. Structure and mechanism of ATP-binding cassette transporters. Philos. Trans. R. Soc. London, Ser. B 2009, 364 (1514), 239–245. 10.1098/rstb.2008.0125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dawson R. J. P.; Hollenstein K.; Locher K. P. Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism. Mol. Microbiol. 2007, 65 (2), 250–257. 10.1111/j.1365-2958.2007.05792.x. [DOI] [PubMed] [Google Scholar]
  4. Shirihai O. S.; Gregory T.; Yu C. N.; Orkin S. H.; Weiss M. J. ABC-me: a novel mitochondrial transporter induced by GATA-1 during erythroid differentiation. EMBO J. 2000, 19 (11), 2492–2502. 10.1093/emboj/19.11.2492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Zhang F.; Hogue D. L.; Liu L.; Fisher C. L.; Hui D.; Childs S.; Ling V. M-ABC2, a new human mitochondrial ATP-binding cassette membrane protein. FEBS Lett. 2000, 478 (1–2), 89–94. 10.1016/S0014-5793(00)01823-8. [DOI] [PubMed] [Google Scholar]
  6. Gonzalez-Ibanez A. M.; Ruiz L. M.; Jensen E.; Echeverria C. A.; Romero V.; Stiles L.; Shirihai O. S.; Elorza A. A. Erythroid Differentiation and Heme Biosynthesis Are Dependent on a Shift in the Balance of Mitochondrial Fusion and Fission Dynamics. Front. Cell Dev. Biol. 2020, 8, 592035. 10.3389/fcell.2020.592035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hyde B. B.; Liesa M.; Elorza A. A.; Qiu W.; Haigh S. E.; Richey L.; Mikkola H. K.; Schlaeger T. M.; Shirihai O. S. The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo. Cell Death Differ. 2012, 19 (7), 1117–1126. 10.1038/cdd.2011.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Martinez M.; Fendley G. A.; Saxberg A. D.; Zoghbi M. E. Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin. PLoS One 2020, 15 (11), e0238754 10.1371/journal.pone.0238754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Shum M.; Shintre C. A.; Althoff T.; Gutierrez V.; Segawa M.; Saxberg A. D.; Martinez M.; Adamson R.; Young M. R.; Faust B.; et al. ABCB10 exports mitochondrial biliverdin, driving metabolic maladaptation in obesity. Sci. Transl. Med. 2021, 13 (594), eabd1869 10.1126/scitranslmed.abd1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Shintre C. A.; Pike A. C.; Li Q.; Kim J. I.; Barr A. J.; Goubin S.; Shrestha L.; Yang J.; Berridge G.; Ross J.; et al. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (24), 9710–9715. 10.1073/pnas.1217042110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dowhan W. Molecular basis for membrane phospholipid diversity: why are there so many lipids?. Annu. Rev. Biochem. 1997, 66, 199–232. 10.1146/annurev.biochem.66.1.199. [DOI] [PubMed] [Google Scholar]
  12. Colbeau A.; Nachbaur J.; Vignais P. M. Enzymac characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta 1971, 249 (2), 462–492. 10.1016/0005-2736(71)90123-4. [DOI] [PubMed] [Google Scholar]
  13. Hovius R.; Lambrechts H.; Nicolay K.; Dekruijff B. Improved Methods to Isolate and Subfractionate Rat-Liver Mitochondria - Lipid-Composition of the Inner and Outer-Membrane. Biochim. Biophys. Acta 1990, 1021 (2), 217–226. 10.1016/0005-2736(90)90036-N. [DOI] [PubMed] [Google Scholar]
  14. Daum G.; Vance J. E. Import of lipids into mitochondria. Prog. Lipid Res. 1997, 36 (2–3), 103–130. 10.1016/S0163-7827(97)00006-4. [DOI] [PubMed] [Google Scholar]
  15. Horvath S. E.; Daum G. Lipids of mitochondria. Prog. Lipid Res. 2013, 52 (4), 590–614. 10.1016/j.plipres.2013.07.002. [DOI] [PubMed] [Google Scholar]
  16. Bottinger L.; Horvath S. E.; Kleinschroth T.; Hunte C.; Daum G.; Pfanner N.; Becker T. Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes. J. Mol. Biol. 2012, 423 (5), 677–686. 10.1016/j.jmb.2012.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Claypool S. M. Cardiolipin, a critical determinant of mitochondrial carrier protein assembly and function. Biochim. Biophys. Acta Biomembr. 2009, 1788 (10), 2059–2068. 10.1016/j.bbamem.2009.04.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Patil V. A.; Fox J. L.; Gohil V. M.; Winge D. R.; Greenberg M. L. Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis. J. Biol. Chem. 2013, 288 (3), 1696–1705. 10.1074/jbc.M112.428938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Bolla J. R.; Agasid M. T.; Mehmood S.; Robinson C. V. Membrane Protein-Lipid Interactions Probed Using Mass Spectrometry. Annu. Rev. Biochem. 2019, 88, 85–111. 10.1146/annurev-biochem-013118-111508. [DOI] [PubMed] [Google Scholar]
  20. Marcoux J.; Wang S. C.; Politis A.; Reading E.; Ma J.; Biggin P. C.; Zhou M.; Tao H.; Zhang Q.; Chang G.; et al. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (24), 9704–9709. 10.1073/pnas.1303888110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laganowsky A.; Reading E.; Hopper J. T. S.; Robinson C. V. Mass spectrometry of intact membrane protein complexes. Nat. Protoc. 2013, 8 (4), 639–651. 10.1038/nprot.2013.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laganowsky A.; Reading E.; Allison T. M.; Ulmschneider M. B.; Degiacomi M. T.; Baldwin A. J.; Robinson C. V. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 2014, 510 (7503), 172–175. 10.1038/nature13419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hilton G. R.; Benesch J. L. Two decades of studying non-covalent biomolecular assemblies by means of electrospray ionization mass spectrometry. J. R. Soc., Interface 2012, 9 (70), 801–816. 10.1098/rsif.2011.0823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Cong X.; Liu Y.; Liu W.; Liang X.; Russell D. H.; Laganowsky A. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry. J. Am. Chem. Soc. 2016, 138 (13), 4346–4349. 10.1021/jacs.6b01771. [DOI] [PubMed] [Google Scholar]
  25. Liu Y.; Locaste C. E.; Liu W.; Poltash M. L.; Russell D. H.; Laganowsky A. Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel. Nat. Commun. 2019, 10, 1352. 10.1038/s41467-019-09333-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gupta K.; Donlan J. A. C.; Hopper J. T. S.; Uzdavinys P.; Landreh M.; Struwe W. B.; Drew D.; Baldwin A. J.; Stansfeld P. J.; Robinson C. V. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 2017, 541 (7637), 421–424. 10.1038/nature20820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Cong X.; Liu Y.; Liu W.; Liang X.; Laganowsky A. Allosteric modulation of protein-protein interactions by individual lipid binding events. Nat. Commun. 2017, 8 (1), 2203. 10.1038/s41467-017-02397-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Patrick J. W.; Boone C. D.; Liu W.; Conover G. M.; Liu Y.; Cong X.; Laganowsky A. Allostery revealed within lipid binding events to membrane proteins. Proc. Natl. Acad. Sci. U.S.A. 2018, 115 (12), 2976–2981. 10.1073/pnas.1719813115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gault J.; Donlan J. A.; Liko I.; Hopper J. T.; Gupta K.; Housden N. G.; Struwe W. B.; Marty M. T.; Mize T.; Bechara C.; et al. High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods 2016, 13 (4), 333–336. 10.1038/nmeth.3771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Scholz J.; Suppmann S. A new single-step protocol for rapid baculovirus-driven protein production in insect cells. BMC Biotechnol. 2017, 17 (1), 83. 10.1186/s12896-017-0400-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Chen P. S.; Toribara T. Y.; Warner H. Microdetermination of Phosphorus. Anal. Chem. 1956, 28 (11), 1756–1758. 10.1021/ac60119a033. [DOI] [Google Scholar]
  32. Marty M. T.; Baldwin A. J.; Marklund E. G.; Hochberg G. K. A.; Benesch J. L. P.; Robinson C. V. Bayesian Deconvolution of Mass and Ion Mobility Spectra: From Binary Interactions to Polydisperse Ensembles. Anal. Chem. 2015, 87 (8), 4370–4376. 10.1021/acs.analchem.5b00140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lanzetta P. A.; Alvarez L. J.; Reinach P. S.; Candia O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 1979, 100 (1), 95–97. 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  34. Reading E.; Liko I.; Allison T. M.; Benesch J. L.; Laganowsky A.; Robinson C. V. The role of the detergent micelle in preserving the structure of membrane proteins in the gas phase. Angew. Chem., Int. Ed. Engl. 2015, 54 (15), 4577–4581. 10.1002/anie.201411622. [DOI] [PubMed] [Google Scholar]
  35. Qiao P.; Schrecke S.; Lyu J.; Zhu Y.; Zhang T.; Benavides A.; Laganowsky A. Insight into the Phospholipid-Binding Preferences of Kir3.4. Biochemistry 2021, 60, 3813–3821. 10.1021/acs.biochem.1c00615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Poltash M. L.; McCabe J. W.; Patrick J. W.; Laganowsky A.; Russell D. H. Development and Evaluation of a Reverse-Entry Ion Source Orbitrap Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2019, 30 (1), 192–198. 10.1007/s13361-018-1976-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Campuzano I. D. G.; Pelegri-O’Day E. M.; Srinivasan N.; Lippens J. L.; Egea P.; Umeda A.; Aral J.; Zhang T.; Laganowsky A.; Netirojjanakul C. High-Throughput Mass Spectrometry for Biopharma: A Universal Modality and Target Independent Analytical Method for Accurate Biomolecule Characterization. J. Am. Soc. Mass Spectrom. 2022, 33 (11), 2191–2198. 10.1021/jasms.2c00138. [DOI] [PubMed] [Google Scholar]
  38. Ree R.; Varland S.; Arnesen T. Spotlight on protein N-terminal acetylation. Exp. Mol. Med. 2018, 50 (7), 1–13. 10.1038/s12276-018-0116-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schrecke S.; Zhu Y.; McCabe J. W.; Bartz M.; Packianathan C.; Zhao M.; Zhou M.; Russell D.; Laganowsky A. Selective regulation of human TRAAK channels by biologically active phospholipids. Nat. Chem. Biol. 2021, 17 (1), 89–95. 10.1038/s41589-020-00659-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Qiao P.; Liu Y.; Zhang T.; Benavides A.; Laganowsky A. Insight into the Selectivity of Kir3.2 toward Phosphatidylinositides. Biochemistry 2020, 59 (22), 2089–2099. 10.1021/acs.biochem.0c00163. [DOI] [PubMed] [Google Scholar]

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