RESUMO
Many biological membranes present an asymmetric lipid distribution between the two leaflets that is known as the transbilayer lipid asymmetry. This asymmetry is essential for cell survival and its loss is related to apoptosis. In mammalian and yeast cells, ATP-dependent transport of lipids to the cytosolic side of the biological membranes, carried out by so-called lipid flippases, contributes to the transbilayer lipid asymmetry. Most of these lipid flippases belong to the P4-ATPase protein family, which is also present in plants. In this review, we summarize the relatively scarce literature concerning the presence of transbilayer lipid asymmetry in different plant cell membranes and revise the potential role of lipid flippases of the P4-ATPase family in generation and/or maintenance of this asymmetry.
Assuntos
Adenosina Trifosfatases , Lipídeos , Animais , Membrana Celular/metabolismo , Adenosina Trifosfatases/metabolismo , Mamíferos/metabolismoRESUMO
Lipoteichoic acids (LTA) are ubiquitous cell wall components of Gram-positive bacteria. In Staphylococcus aureus LTA are composed of a polymer with 1,3-linked glycerol phosphate repeating units anchored to the plasma membrane. The anchor molecule is a lipid-linked disaccharide (anchor-LLD) synthesized at the cytoplasmic leaflet of the membrane. The anchor lipid becomes accessible at the outer leaflet of the membrane after the flippase LtaA catalyzes translocation. Recently we have elucidated the structure of LtaA using vapor diffusion X-ray crystallography and in situ annealing. We were able to obtain LtaA crystals after optimization of purification protocols that led to stabilization of LtaA isolated in detergent micelles. Here we report a protocol that describes the purification, stabilization, crystallization, and data collection strategies carried out to determine the structure of LtaA. We highlight key points that can be used to determine crystal structures of other membrane proteins.
Assuntos
Bioquímica/métodos , Proteínas de Transporte , Lipopolissacarídeos/metabolismo , Proteínas de Membrana , Renaturação Proteica , Ácidos Teicoicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Fenômenos Bioquímicos , Transporte Biológico , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Cristalização , Cristalografia por Raios X , Detergentes/química , Detergentes/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Micelas , Estabilidade Proteica , Staphylococcus aureus/química , Staphylococcus aureus/metabolismoRESUMO
ATP-dependent phospholipid flippase activity crucial for generating lipid asymmetry was first detected in red blood cell (RBC) membranes, but the P4-ATPases responsible have not been directly determined. Using affinity-based MS, we show that ATP11C is the only abundant P4-ATPase phospholipid flippase in human RBCs, whereas ATP11C and ATP8A1 are the major P4-ATPases in mouse RBCs. We also found that ATP11A and ATP11B are present at low levels. Mutations in the gene encoding ATP11C are responsible for blood and liver disorders, but the disease mechanisms are not known. Using heterologous expression, we show that the T415N substitution in the phosphorylation motif of ATP11C, responsible for congenital hemolytic anemia, reduces ATP11C expression, increases retention in the endoplasmic reticulum, and decreases ATPase activity by 61% relative to WT ATP11C. The I355K substitution in the transmembrane domain associated with cholestasis and anemia in mice was expressed at WT levels and trafficked to the plasma membrane but was devoid of activity. We conclude that the T415N variant causes significant protein misfolding, resulting in low protein expression, cellular mislocalization, and reduced functional activity. In contrast, the I355K variant folds and traffics normally but lacks key contacts required for activity. We propose that the loss in ATP11C phospholipid flippase activity coupled with phospholipid scramblase activity results in the exposure of phosphatidylserine on the surface of RBCs, decreasing RBC survival and resulting in anemia.
Assuntos
Adenosina Trifosfatases/metabolismo , Eritrócitos/enzimologia , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/genética , Animais , Membrana Eritrocítica/enzimologia , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosforilação , Dobramento de ProteínaRESUMO
Lipid flippases are integral membrane proteins that use ATP hydrolysis to power the generation of phospholipid asymmetry between the two leaflets of biological membranes, a process essential for cell survival. Although the first report of a plant lipid flippase was published in 2000, progress in the field has been slow, partially due to the high level of redundancy in this gene family. However, recently an increasing number of reports have examined the physiological function of lipid flippases, mainly in Arabidopsis thaliana. In this review we aim to summarize recent findings on the physiological relevance of lipid flippases in plant adaptation to a changing environment and caution against misinterpretation of pleiotropic effects in genetic studies of flippases.
Assuntos
Adenosina Trifosfatases , Fosfolipídeos , Membrana Celular , Proteínas de Membrana , FenótipoRESUMO
In all eukaryotic cells, P4 ATPases, also named phospholipid flippases, generate phospholipid asymmetry across biological membranes. This process is essential for cell survival, as it is required for vesicle budding and fusion in the secretory pathway. Several P4 ATPase isoforms can be identified in all sequenced eukaryotic genomes, but their evolution and interrelationships are poorly described. In this study, we conducted a thorough phylogenetic analysis of P4 ATPases in all major eukaryotic super-groups and found that they can be divided into three distinct families, P4A, P4B and P4C ATPases, all of which have an ancient origin. While P4B ATPases have been lost in plants, P4A ATPases are present in all eukaryotic super-groups. P4C ATPases form an intermediate group between the other two but appear to share a common origin with P4A ATPases. Sequence motifs unique to P4 ATPases are situated in the basal ATP hydrolyzing machinery. In addition, no clear signature motifs within P4 ATPase subgroups were found that could be related to lipid specificity, likely pointing to an elaborate transport mechanism in which different amino acid residue combinations in these pumps can result in recognition of the same substrate.
Assuntos
Adenosina Trifosfatases/metabolismo , Evolução Biológica , Terminologia como Assunto , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Domínio Catalítico , Citoplasma/enzimologia , Células Eucarióticas/enzimologia , Filogenia , Homologia de Sequência de AminoácidosRESUMO
Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions.