University of Groningen
Insights into the transport mechanism of energy-coupling factor transporters
Stanek, Weronika Karolina
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CHAPTER 6
Effect of lipids on the ECF transporters activity
Weronika K Stanek, Shahid Mehmood, Joris MH Goudsmits, Dirk Jan Slotboom
ABSTRACT
Membrane proteins are embedded in the lipid bilayer and often have developed interactions with specific lipids. The activity and stability of membrane proteins in detergent or in non-native lipids may vary from their physiological environment. Here, we started identification of lipids affecting the activity of ECF transporters in proteoliposomes. We identified lipids that are tightly bound to transporters and measured transport activity in proteoliposomes consisting of different lipid mixtures. We conclude that ECF transporters interact with non-bilayer forming lipids such as phosphatidylethanolamine, and that their activity in enhanced in mixtures containing such lipids.
INTRODUCTION
The cell’s cytoplasm is surrounded by a semipermeable membrane. It consists of a lipid bilayer and embedded membrane proteins. Membrane proteins evolved to be part of the membranes, and lipids are necessary for their stability and activity.1,2 Due to differences
in lipid components of membranes from different organisms individual membrane proteins may have evolved to interact with specific components, and therefore the relation between membrane protein function and lipid dependency is crucial to understand.3
Energy-Coupling Factor (ECF) transporters are ATP-Binding Cassette (ABC) type transporters residing in the plasma membrane of prokaryotes. They consist of two cytoplasmic, soluble ATPases (EcfA and EcfA’) and two transmembrane proteins (EcfT and S-component). The two transmembrane proteins have different fold and role in the protein activity. EcfT is a component that connects to all other subunits and transmits motion of the ATPases to the S-component. EcfT possesses both transmembrane and peripheral, cytosolic helices. The S-components are entirely embedded in the membrane,4,5 with small extra membranous loops.
They are responsible for the substrate binding and translocation through the lipid bilayer. The mechanism of transport in ECF transporters requires S-components to be dynamic. They use a unique toppling mechanism, which involves large rotation motions in the membrane.6,7 In
addition, they can associate with and dissociate from the energizing module (EcfAA’T), likely as part of the catalytic cycle.8–10 These observations raise questions on the effect of the lipid
composition on transport activity, and possible requirements for specific lipids. As the lipid bilayer allows for lateral diffusion of its components, lipids and proteins,11 association and
dissociation of S-components is expected to be possible, but a challenge could be the change in exposed surfaces, which may match different membrane thicknesses. The interfaces are covered by the interacting subunits in the full complex transporter and are exposed to the lipids when S-component dissociate.
Here, we investigated what lipids are associated with ECF transporters. Using Mass Spectrometry, it was possible to detect lipids co-purified with the S-components and with the full complex. Co-purification of a lipid indicates its strong interaction with the protein and could be indication that the lipid affects activity. Simultaneously, an attempt was made to identify the lipid composition of liposomes required for high transport activity. Knowledge of the optimal environment for full complex ECF transporter and solitary S-components may be used to increase protein stability during purification which has been problematic. Moreover, knowledge of lipid interactions with S-components can bring a new insight for the toppling mechanism.
METHODS
Protein overexpression, purification and reconstitution into proteoliposomes
Full complexes ECF-PanT or ECF-FolT2 from Lactobacillus delbrueckii were produced in Escherichia coli MC1061 from expression plasmids p2BAD MCS1 10HisTEV ecfAA’T, MCS2 panT-StrepII or p2BAD MCS1 10HisTEV ecfAA’T, MCS2 folT2-StrepII, respectively (Chapter 3). Protein purification from membranes was performed with n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) as described in Swier et al.7 and Chapter 3.
Solitary ThiT from Lactococcus lactis was expressed in L.lactis NZ9000 from pNZ8048 with thiT gene tagged with a sequence coding for an N-terminal 8xHis-tag.12 The protein
was overexpressed in M17 broth (Difco) supplemented with 1% (v/v) glucose and 5 µg/ml chloramphenicol at 30°C. The expression was induced with 0,1% (v/v) supernatant from the nisin A producing strain at OD600 of 1.2.13 The cells were harvested by centrifugation (15
min, 6268 × g at 4°C) after 3 hours of expression. A detailed procedure of membrane vesicles preparation and ThiT purification with n-Decyl-β-D-Maltopyranoside (DM, Anatrace) is described in Swier et al.14
Solitary BioY from L.lactis was expressed according to 15. The protocols for expression and
membrane vesicles isolation were identical as for solitary ThiT. The purification with maltose neoprentyl glycerol (MNG-3, Anatrace) was performed according to protocol described in Berntsson et al.15
Full complex ECF transporters were reconstituted to the Triton X-100 destabilized liposomes with the detergents removed with BioBeads.16 The protein to lipid ratio for uptake
experiments was 1:1000 (w/w). Different lipid mixtures were made and listed in Table 1. The E.coli polar lipids were obtained by washing E.coli total lipid extract (Avanti) with acetone and diethylether. Further, washed extract was supplemented with L-α-phosphatidylcholine from egg (Avanti). The synthetic lipids formulations were created by mixing chloroform solutions of lipids (Avanti) in the ratios stated in the Table 1. We compared our standard lipid composition (E.coli polar lipids with 25% egg PC) with different synthetic lipids. For formulations 3.7:5:1.3 and 2:6:2 both 1,2-dioleoyl (DO) and 1-palmitoyl-2-oleoyl (PO) forms of lipids were used. The rest of lipid mixtures (4:3:3, ECF1 and ECF2) were tested only in DO form of lipids.
Table 1 Lipid compositions used for activity tests in ECF-PanT. Lipid E.coli total extract phospholipids extract E.coli polar lipids extract E.coli polar lipids+ egg PC 4:3:3 3.7:5:1.3 2:6:2 ECF1 ECF2 PE 57.5 67.0 50.3 30 50 60 55 45 PG 15.1 23.2 17.4 30 13 20 15 15 PC 0 0 24.5 40 37 20 20 20 CL 9.8 9.8 7.4 0 0 0 10 20 SM 0 0 0.4 0 0 0 0 0 unknown 17.6 0 0 0 0 0 0 0
*PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PC (phosphatidylcholine), CL (cardiolipin), SM (sphingomyelin). The numbers indicate the percentage of each lipid present (in weight).
Mass spectrometry of lipids co-purified with protein
solitary ThiT and BioY and full complex ECF-PanT and ECF-FolT2 is described by Mehmood et al.17
Radiolabeled substrate uptake assays
Uptake assays were performed in ECF-PanT proteoliposomes loaded with Mg-ATP or Mg-ADP. As a substrate D-[2,3-3H]pantothenic acid sodium salt (American Radiolabeled
Chemicals) was used. Assays were performed as described in Chapter 3.
RESULTS
Lipids co-purified with full complex ECF transporters
We performed an analysis of the lipids co-purifying with the ECF-PanT and ECF-FolT2. Both proteins originate from L. delbrueckii, and are composed of identical EcfAA’T modules but different S-components. Each complex was expressed in E.coli and purified using Ni-affinity and size-exclusion chromatography using the detergent DDM. After purification, the proteins were subjected to tryptic digestion with the aim to liberate bound lipids and facilitate lipid analysis. HPLC was used for lipid separation, and mass spectrometry for identification. Lipidomics of both transporters showed the presence of two different classes of lipids, Phosphatidyl Ethanolamine (PE) and Phosphatidyl Glycerol (PG) (Figure 1). For each class we found specific lipids with acyl chains 16:0/16:1 and 16:1/18:1. The ECF-PanT sample contained less lipids compared to the ECF-FolT2 sample. The difference in lipid amount may indicate that lipid binding is specific to different S-components, but it should be noted that the experiment has been performed only once, and random errors in the analysis could also account for the differences in amounts of co-purified lipids.
Figure 1 Chromatogram from reversed-phase chromatography representing the abundance of detected lipids in ECF-FolT2. The phosphatidylethanolamine (PE) is shown in blue (16:0/16:1) and red (16:1/18:1), phosphatidylglycerol (PG) is shown in green (16:0/16:1) and purple (16:1/18:1)
Lipids co-purified with the solitary S-components
S-components are not permanently associated with an ECF module and they can dissociate from the ECF module during the catalytic cycle. Moreover, they can be upregulated to much
higher levels that the ECF module. Therefore, we also analyzed the lipid associated with solitary S-components. We chose the S-components BioY and ThiT from L. lactis for the analysis because they can be purified in large quantities from the native host organism (rather than heterologously in E.coli, which is used as expression host for the full complexes). Therefore, the S-components are exposed only to its natural lipid environment which makes the results more relevant. Lipidomic analysis of BioY revealed that PG, cardiolipins, DG (Dioleoyl glycerol), and MGDG (monogalactosyldiacylglycerol) were co-purified with the protein (Figure 2). In case of ThiT, we found PG, DGDG (digalactosyldiacylglycerol) and cardiolipin (Figure 3). In both cases the most abundant lipid was PG with additional smaller populations of cardiolipins. The presence of DG, a precursor for biosynthesis of some phospholipids, could also be a hydrolysis product of PG.18 The two samples showed
co-purification of trace amounts of the glycolipids (di- and mono-galactosyldiacylglycerol, DGDG and MGDG, respectively).
Figure 2 Chromatogram and mass spectrum of lipids co-purified with BioY determined by reversed-phase HPLC connected with tandem mass spectrometer. Lipids were manually identified based on their fragmentation pattern.
Figure 3 Chromatogram and mass spectrum of lipids co-purified with ThiT determined by reversed-phase HPLC connected with mass spectrometer.
Influence of lipid composition of liposomes on ECF-PanT activity
We tested the effect of the lipid composition on the activity of ECF transporter. Wild type ECF-PanT was reconstituted into proteoliposomes composed of different lipid mixtures (Table 1), and transport activity was assayed by uptake assays using radiolabeled pantothenate. We tested the transport activity in liposomes composed of E.coli polar lipids, which is a complex and partially undefined mixture, supplemented with egg PC, and in liposomes consisting of various mixtures of synthetic lipids.The highest rates of pantothenate uptake in the lumen of proteoliposomes were observed for liposomes composed of E.coli polar lipids supplemented with egg PC. The results of the uptakes in liposomes with defined lipid mixtures show that ECF-PanT is more active in proteoliposomes with higher PE content (Figure 4). Because we compensated the changes in the PE content by adjusting the amount of PC, it cannot be excluded that high concentrations of PC negatively affect activity. However, effects of PC seem less likely, as the the highest activity is found in the E.coli lipid mixture supplemented with 25% PC. In the tested compositions, the PG content was not systematically changed which precludes conclusions on the potential role of PG for ECF-PanT activity. Also, the influence of cardiolipins could not be determined unambiguously. The pool of cardiolipins was added in place of PE and PG. Therefore the lowered PE content may have masked the effect of cardiolipins.
Figure 4 Effect of lipids on the ECF-PanT activity. Transport activity of ECF-PanT in different proteoliposomes. Abbreviations: E.coli lipids – E.coli polar lipids + egg PC proteoliposomes; 4:3:3 – 40% PC, 30% PE, 30%PG; 2:6:2 – 20% PC, 60% PE, 20% PG; 3.7:5:1.3 – 37% PC, 50% PE, 13% PG; ECF1 – 20% PC, 55% PE, 15% PG, 10% CL; ECF2 – 20% PC, 45% PE, 15% PG, 20% CL; E.coli ADP - E.coli polar lipids + egg PC proteoliposomes loaded with Mg-ADP (negative control). DO refers to 1,2-dioleoyl lipids and PO to 1-palmitoyl-2-oleoyl variants.
DISCUSSION
Membrane proteins need lipids to cover their hydrophobic surfaces from the aqueous environment. However, some proteins are more demanding for the lipid composition or may require the binding of a specific for their activity. It was shown for many membrane proteins that they require specific lipidic environment for optimal functioning.1,19–21 These protein may
require a specific membrane fluidity22–24, charge19,25,26 or just a specific lipid.27
In the study presented here we have begun to identify lipids associated with ECF transporters, which may have an impact on stability or activity of the full transporter complexes and solitary S-components. Lipidomic analysis of ECF-PanT and ECF-FolT2 revealed the most abundant lipids in the expression host E.coli (PE and PG)28,29 were also co-purified with the proteins.
Interestingly, we observed differences in lipids abundance between FolT2 and ECF-PanT. In ECF-PanT we detected a smaller amount of co-purified lipids that in ECF-FolT2. Additionally, solitary PanT was highly unstable when purified but addition of lipids during purification improved to some extent the protein’s stability (Chapter 3). Combined, these results suggest that S-components have pronounced effects on the lipid interactions by ECF transporters. Therefore, we also analyzed lipids associated with solitary S-components. In S-component samples that has been produced in the native host L.lactis, PG and cardiolipins were the most abundant lipids co-purified. In the BioY sample MGDG was detected. MGDG is a glycolipid with similar non-bilayer properties as POPE.30,31 MGDG is not present in E.
coli, which may explain why PE was associated with the full complex FolT2 and ECF-PanT. The S-component ThiT was also produced in L. lactis, but in this case the glycolipid DGDG was co-purified instead of MDGD. MGDG and DGDG have galactose moiety linked to the diacylglycerol but because of the amount of attached sugars they have different surface behavior.32 It is not clear if the properties of both glycolipids have similar functional effects
in both proteins.
We also tested how different lipid compositions in liposomes influence the pantothenate transport activity of ECF-PanT reconstituted in liposomes. Our main conclusion is that ECF transporters require high content of non-bilayer lipids like PE. This conclusion is in agreement with observations that many membrane proteins exhibit higher activity in bilayers enriched with PE.30,33–35
In the current study we did not have an opportunity to extend the investigation of lipid influence on transport activity of ECF transporters. It is tempting to speculate on the influence of glycolipids on the stability or toppling of S-components. Further experiments need to be performed both on full complexes and solitary S-components to elucidate the role of the lipid environment in ECF transporter function.
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