Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders.

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, for example in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase.

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na+/K+-ATPase or Ca2+-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.

Sarcolipin alters SERCA1a interdomain communication by impairing binding of both calcium and ATP.

Sarcolipin (SLN), a single-spanning membrane protein, is a regulator of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1a). Chemically synthesized SLN, palmitoylated or not (pSLN or SLN), and recombinant wild-type rabbit SERCA1a expressed in S. cerevisiae design experimental conditions that provide a deeper understanding of the functional role of SLN on the regulation of SERCA1a. Our data show that chemically synthesized SLN interacts with recombinant SERCA1a, with calcium-deprived E2 state as well as with calcium-bound E1 state. This interaction hampers the binding of calcium in agreement with published data. Unexpectedly, SLN has also an allosteric effect on SERCA1a transport activity by impairing the binding of ATP. Our results reveal that SLN significantly slows down the E2 to Ca2.E1 transition of SERCA1a while it affects neither phosphorylation nor dephosphorylation. Comparison with chemically synthesized SLN deprived of acylation demonstrates that palmitoylation is not necessary for either inhibition or association with SERCA1a. However, it has a small but statistically significant effect on SERCA1a phosphorylation when various ratios of SLN-SERCA1a or pSLN-SERCA1a are tested.

ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 ß-subunit.

Gene targeting approaches have demonstrated the essential role for the malaria parasite of membrane transport proteins involved in lipid transport and in the maintenance of membrane lipid asymmetry, representing emerging oportunites for therapeutical intervention. This is the case of ATP2, a Plasmodium-encoded 4 P-type ATPase (P4-ATPase or lipid flippase), whose activity is completely irreplaceable during the asexual stages of the parasite. Moreover, a recent chemogenomic study has situated ATP2 as the possible target of two antimalarial drug candidates. In eukaryotes, P4-ATPases assure the asymmetric phospholipid distribution in membranes by translocating phospholipids from the outer to the inner leaflet. In this work, we have used a recombinantly-produced P. chabaudi ATP2 (PcATP2), to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 associates with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A. Purified PcATP2/PcCdc50B complex displays ATPase activity in the presence of either phosphatidylserine or phosphatidylethanolamine. In addition, this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work describes the first biochemical characterization of a Plasmodium lipid flippase, a first step towards the understanding of the essential physiological role of this transporter and towards its validation as a potential antimalarial drug target.

The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.

Chemical Synthesis of Native S-Palmitoylated Membrane Proteins through Removable-Backbone-Modification-Assisted Ser/Thr Ligation.

The preparation of native S-palmitoylated (S-palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S-palm membrane proteins by removable-backbone-modification-assisted Ser/Thr ligation (RBMGABA -assisted STL). This method involves two critical steps: 1) synthesis of S-palm peptides by a new γ-aminobutyric acid based RBM (RBMGABA ) strategy, and 2) ligation of the S-palm RBM-modified peptides to give the desired S-palm product by the STL method. The utility of the RBMGABA -assisted STL method was demonstrated by the synthesis of rabbit S-palm sarcolipin (SLN) and S-palm matrix-2 (M2) ion channel. The synthesis of S-palm membrane proteins highlights the importance of developing non-NCL methods for chemical protein synthesis.

Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins.

The photoactive Orange Carotenoid Protein (OCP) photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain is the active part of the protein, and the C-terminal domain regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins [HCPs]), paralogs of the N-terminal domain of OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized, and they proved to be very good singlet oxygen quenchers. When synthesized in Escherichia coli or Synechocystis PCC 6803, CTDHs formed dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo-CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable to do so. HCPs need the presence of CTDH to become holo-proteins. We propose that, in cyanobacteria, the CTDHs are carotenoid donors to HCPs.

High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions.

P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after Arg104 and after Arg 1290, resulting in a homogeneous sample lacking 104 and 65 residues from its N and C termini, respectively. Removal of the 1291-1302-amino acid region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, whereas the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p-Cdc50p flippase follows a multistep mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in the trans-Golgi network, followed by full activation by PI4P.

Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport.

Phospholipid flippases are key regulators of transbilayer lipid asymmetry in eukaryotic cell membranes, critical to many trafficking and signaling pathways. P4-ATPases, in particular, are responsible for the uphill transport of phospholipids from the exoplasmic to the cytosolic leaflet of the plasma membrane, as well as membranes of the late secretory/endocytic pathways, thereby establishing transbilayer asymmetry. Recent studies combining cell biology and biochemical approaches have improved our understanding of the path taken by lipids through P4-ATPases. Additionally, identification of several protein families catalyzing phospholipid 'scrambling', i.e. disruption of phospholipid asymmetry through energy-independent bi-directional phospholipid transport, as well as the recent report of the structure of such a scramblase, opens the way to a deeper characterization of their mechanism of action. Here, we discuss the molecular nature of the mechanism by which lipids may 'flip' across membranes, with an emphasis on active lipid transport catalyzed by P4-ATPases. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex.

Structural and functional characterization of integral membrane proteins requires milligram amounts of purified sample. Unless the protein you are studying is abundant in native membranes, it will be critical to overexpress the protein of interest in a homologous or heterologous way, and in sufficient quantities for further purification. The situation may become even more complicated if you chose to investigate the structure and function of a complex of two or more membrane proteins. Here, we describe the overexpression of a yeast lipid flippase complex, namely the P4-ATPase Drs2p and its associated subunit Cdc50p, in a coordinated manner. Moreover, we can take advantage of the fact that P4-ATPases, like most other P-type ATPases, form an acid-stable phosphorylated intermediate, to verify that the expressed complex is functional.

Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion.

UNLABELLED: In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE: HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.

A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate.

P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼ 1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1:1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

S-palmitoylation and s-oleoylation of rabbit and pig sarcolipin.

Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.

SERCA mutant E309Q binds two Ca(2+) ions but adopts a catalytically incompetent conformation.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) couples ATP hydrolysis to transport of Ca(2+). This directed energy transfer requires cross-talk between the two Ca(2+) sites and the phosphorylation site over 50 Å distance. We have addressed the mechano-structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu(309) contributes to Ca(2+) coordination at site II, and a consensus has been that E309Q only binds Ca(2+) at site I. The crystal structure of E309Q in the presence of Ca(2+) and an ATP analogue, however, reveals two occupied Ca(2+) sites of a non-catalytic Ca2E1 state. Ca(2+) is bound with micromolar affinity by both Ca(2+) sites in E309Q, but without cooperativity. The Ca(2+)-bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring a shift of transmembrane segment M1 into an 'up and kinked position'. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca(2+) site II.

Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy.

Recombinant Ca(2+)-ATPase was expressed in Saccharomyces cerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200 µg of ~ 70% pure recombinant sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 1a (SERCA1a) from a 6-L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca2 E1P and Ca2 E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca2 E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.

Phosphatidylserine stimulation of Drs2p·Cdc50p lipid translocase dephosphorylation is controlled by phosphatidylinositol-4-phosphate.

Here, Drs2p, a yeast lipid translocase that belongs to the family of P(4)-type ATPases, was overexpressed in the yeast Saccharomyces cerevisiae together with Cdc50p, its glycosylated partner, as a result of the design of a novel co-expression vector. The resulting high yield allowed us, using crude membranes or detergent-solubilized membranes, to measure the formation from [γ-(32)P]ATP of a (32)P-labeled transient phosphoenzyme at the catalytic site of Drs2p. Formation of this phosphoenzyme could be detected only if Cdc50p was co-expressed with Drs2p but was not dependent on full glycosylation of Cdc50p. It was inhibited by orthovanadate and fluoride compounds. In crude membranes, the phosphoenzyme formed at steady state at 4 °C displayed ADP-insensitive but temperature-sensitive decay. Solubilizing concentrations of dodecyl maltoside left this decay rate almost unaltered, whereas several other detergents accelerated it. Unexpectedly, the dephosphorylation rate for the solubilized Drs2p·Cdc50p complex was inhibited by the addition of phosphatidylserine. Phosphatidylserine exerted its anticipated accelerating effect on the dephosphorylation of Drs2p·Cdc50p complex only in the additional presence of phosphatidylinositol-4-phosphate. These results explain why phosphatidylinositol-4-phosphate tightly controls Drs2p-catalyzed lipid transport and establish the functional relevance of the Drs2p·Cdc50p complex overexpressed here.

The Plasmodium falciparum Ca(2+)-ATPase PfATP6: insensitive to artemisinin, but a potential drug target.

The disease malaria, caused by the parasite Plasmodium falciparum, remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca2+-ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139 kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca2+-binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum.

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: low concentrations of HNE open protein-mediated leaks in the membrane.

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca(2+) transport ability of SERCA1a, the Ca(2+) pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca(2+) binding to the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys(515) within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca(2+) and the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNE-treated SR vesicles to Ca(2+), an increase in permeability that did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150-160kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca(2+) leakage pathways might be involved in the general toxic effects of HNE.