Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

Gantzel, Rasmus Hvidbjerg; Mogensen, Louise S.; Mikkelsen, Stine A.; Vilsen, Bente; Molday, Robert S.; Vestergaard, Anna L.; Andersen, Jens Peter

doi:10.1038/s41598-017-10741-z

Cited by 22 publications

(17 citation statements)

References 25 publications

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“…port in a human phosphatidylserine-translocating P4-ATPase, ATP8A2 (35), although the QQ motif was not directly examined. These studies collectively indicate that this GA/QQ motif in TM1 is an important component of GlcCer/PS recognition but does not contribute to PE/PC selection.…”

Section: Discovery Of Yeast and Human Glucosylceramide P4-atpasesmentioning

confidence: 99%

Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs

Roland

Naito

Best

et al. 2019

Journal of Biological Chemistry

127

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Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genomewide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover, ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer bearing an sn2 acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis. Phospholipid flippases in the P4-ATPase family establish and repair the phospholipid asymmetry of cellular membranes and facilitate import of phospholipid from extracellular sources (1). These enzymes are integral membrane proteins that harness energy from ATP catalysis to translocate lipids from the exofacial leaflet to the cytofacial leaflet of cellular membranes. There are 14 distinct P4-ATPases in humans with important differences in tissue-specific expression, subcellular localization, and substrate specificity (2). These differences in P4-ATPase localization and enzymology are important for coordinating physiological processes, and their impairment results in diverse pathologies (3). For example, the P4-ATPase ATP8B1 transports phosphatidylcholine at the apical membrane of canalicular hepatocytes (4), and mutations in the ATP8B1 gene lead to membrane damage and hepatic cholestasis (3, 5). Conversely, ATP8A2 translocates phosphatidylserine within the central nervous system, and mutations in ATP8A2 cause cerebellar ataxia mental retardation and disequilibrium syndrome in humans and motor neuron degeneration in mice (6-8). Thus, defining the substrate specificity of the P4-ATPases is crucial to understanding their role in disease. Genome-wide association (GWA) 3 studies have found that SNPs in the human P4-ATPase genes ATP10A and ATP10D are strongly linked with meta...

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Section: Discovery Of Yeast and Human Glucosylceramide P4-atpasesmentioning

confidence: 99%

Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs

Roland

Naito

Best

et al. 2019

Journal of Biological Chemistry

127

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“…Although site-directed mutagenesis and structural modeling studies have provided useful models to describe a lipid translocation pathway in P4-ATPases (7)(8)(9)(10)(11), central aspects of the transport pathway and flipping mechanism remain elusive, including the electrogenic properties of the flippases. Electrogenicity is likewise an unexplored feature for other ATP-dependent lipid translocating systems in biological membranes such as ABC transporters.…”

Section: Significancementioning

confidence: 99%

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic

Tadini‐Buoninsegni

Mikkelsen

Mogensen

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.

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“…The transport mechanism of P4-ATPases is the subject of intensive research, and various models have been proposed for the phospholipid translocation pathway in P4-ATPases [ 103 , 104 , 105 , 106 , 107 ]. The recent atomic resolution structures of yeast and human P4-ATPases have provided valuable information on different conformational states in the flippase transport cycle, which is depicted by the Albers–Post or E 1 –E 2 scheme commonly used to describe the mechanism of ion transporting P2-type ATPases.…”

Section: P-type Atpases Investigated On Solid Supported Membranesmentioning

confidence: 99%

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

Tadini‐Buoninsegni

2020

Molecules

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P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca2+-ATPase, mammalian Cu+-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

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Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

Cited by 22 publications

References 25 publications

Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs

Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

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