RESUMO
Toxoplasma gondii is an obligate, intracellular apicomplexan protozoan parasite of both humans and animals that can cause fetal damage and abortion and severe disease in the immunosuppressed. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localization and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2. Interestingly, we observed that while TgCerS1 was a fully functional orthologue of the yeast ceramide synthase (Lag1p) capable of catalyzing the conversion of sphinganine to ceramide, in contrast TgCerS2 was catalytically inactive. Furthermore, genomic deletion of TgCerS1 using CRISPR/Cas-9 led to viable but slow-growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of TgCerS2 was only accessible utilizing the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this "pseudo" ceramide synthase, TgCerS2, has a considerably greater role in parasite fitness than its catalytically active orthologue (TgCerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa may have arisen through gene duplication. However, in the Apicomplexa the duplicated copy is hypothesized to have subsequently evolved into a non-functional "pseudo" ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites.
Assuntos
Parasitos , Toxoplasma , Humanos , Animais , Toxoplasma/genética , Duplicação Gênica , Filogenia , Esfingolipídeos , Ceramidas/genética , Proteínas de Protozoários/genéticaRESUMO
Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.
Assuntos
Biocatálise , Retículo Endoplasmático/metabolismo , Fosfatidil-N-Metiletanolamina N-Metiltransferase/química , Fosfatidil-N-Metiletanolamina N-Metiltransferase/metabolismo , Fosfatidiletanolamina N-Metiltransferase/química , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Humanos , Modelos Biológicos , Mutação/genética , Fosfatidil-N-Metiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
Assuntos
Domínio Catalítico , Mutagênese Sítio-Dirigida , Esfingolipídeos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Domínios Proteicos , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.
Assuntos
Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Engenharia de Proteínas , Esfingomielinas/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Sistema Livre de Células , Química Click , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Células HeLa , Humanos , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Esfingomielinas/genética , Transferases (Outros Grupos de Fosfato Substituídos)/químicaRESUMO
Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.
Assuntos
Biocatálise , Esfingomielinas/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Encéfalo/citologia , Encéfalo/enzimologia , Encéfalo/metabolismo , Domínio Catalítico , Sobrevivência Celular , Ativação Enzimática , Éxons/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Mutação Puntual , Transporte Proteico , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
We report short ceramide analogs that can be activated with light and further functionalized using azide-alkyne click chemistry. These molecules, termed scaCers, exhibit increased cell permeability compared to their long-chain analogs as demonstrated using mass spectrometry and imaging. Notably, scaCers enable optical control of apoptosis, which is not observed with long-chain variants. Additionally, they function as photoswitchable substrates for sphingomyelin synthase 2 (SMS2), exhibiting inverted light-dependence compared to their extended analogs.
Assuntos
Apoptose/efeitos da radiação , Ceramidas/química , Fármacos Fotossensibilizantes/química , Alcinos/química , Azidas/química , Permeabilidade da Membrana Celular , Ceramidas/metabolismo , Química Click , Células HeLa , Humanos , Processos Fotoquímicos , Relação Estrutura-Atividade , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
In the formation of COPI vesicles, interactions take place between the coat protein coatomer and membrane proteins: either cargo proteins for retrieval to the endoplasmic reticulum (ER) or proteins that cycle between the ER and the Golgi. While the binding sites on coatomer for ER residents have been characterized, how cycling proteins bind to the COPI coat is still not clear. In order to understand at a molecular level the mechanism of uptake of such proteins, we have investigated the binding to coatomer of p24 proteins as examples of cycling proteins as well as that of ER-resident cargos. The p24 proteins required dimerization to interact with coatomer at two independent binding sites in gamma-COP. In contrast, ER-resident cargos bind to coatomer as monomers and to sites other than gamma-COP. The COPI coat therefore discriminates between p24 proteins and ER-resident proteins by differential binding involving distinct subunits.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Transporte/metabolismo , Proteína Coatomer/metabolismo , Retículo Endoplasmático/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteína Coatomer/química , Proteína Coatomer/genética , Dimerização , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de ProteínaRESUMO
Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision. Here, we report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells. Combining atomic force microscopy on model bilayers with metabolic tracing studies in cells, we demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. Our findings disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light.
Assuntos
Ceramidas/metabolismo , Ceramidas/efeitos da radiação , Luz , Esfingolipídeos/biossíntese , Misturas Complexas , Glucosiltransferases/metabolismo , Células HeLa , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Leveduras/enzimologiaRESUMO
Mechanisms leading to osteoporosis are incompletely understood. Genetic disorders with skeletal fragility provide insight into metabolic pathways contributing to bone strength. We evaluated 6 families with rare skeletal phenotypes and osteoporosis by next-generation sequencing. In all the families, we identified a heterozygous variant in SGMS2, a gene prominently expressed in cortical bone and encoding the plasma membrane-resident sphingomyelin synthase SMS2. Four unrelated families shared the same nonsense variant, c.148C>T (p.Arg50*), whereas the other families had a missense variant, c.185T>G (p.Ile62Ser) or c.191T>G (p.Met64Arg). Subjects with p.Arg50* presented with childhood-onset osteoporosis with or without cranial sclerosis. Patients with p.Ile62Ser or p.Met64Arg had a more severe presentation, with neonatal fractures, severe short stature, and spondylometaphyseal dysplasia. Several subjects had experienced peripheral facial nerve palsy or other neurological manifestations. Bone biopsies showed markedly altered bone material characteristics, including defective bone mineralization. Osteoclast formation and function in vitro was normal. While the p.Arg50* mutation yielded a catalytically inactive enzyme, p.Ile62Ser and p.Met64Arg each enhanced the rate of de novo sphingomyelin production by blocking export of a functional enzyme from the endoplasmic reticulum. SGMS2 pathogenic variants underlie a spectrum of skeletal conditions, ranging from isolated osteoporosis to complex skeletal dysplasia, suggesting a critical role for plasma membrane-bound sphingomyelin metabolism in skeletal homeostasis.
Assuntos
Calcificação Fisiológica/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Osteocondrodisplasias/genética , Osteoporose/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Adulto , Idade de Início , Idoso de 80 Anos ou mais , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação de Sentido Incorreto , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/patologia , Osteoporose/diagnóstico , Osteoporose/patologia , Linhagem , Adulto JovemRESUMO
Ceramides are essential precursors of sphingolipids with a dual role as mediators of apoptotic cell death. Previous work revealed that the ER-resident ceramide phosphoethanolamine (CPE) synthase SMSr/SAMD8 is a suppressor of ceramide-mediated apoptosis in cultured cells. Anti-apoptotic activity of SMSr requires a catalytically active enzyme but also relies on the enzyme's N-terminal sterile α-motif or SAM domain. Here, we demonstrate that SMSr itself is a target of the apoptotic machinery. Treatment of cells with staurosporine or the death receptor ligand FasL triggers caspase-mediated cleavage of SMSr at a conserved aspartate located downstream of the enzyme's SAM domain and upstream of its first membrane span. Taking advantage of reconstitution experiments with SMSr produced in a cell-free expression system, specific caspase-inhibitors and gene silencing approaches, we show that SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Huntington's and Alzheimer's diseases. Our findings underscore a role of SMSr as negative regulator of ceramide-induced cell death and, in view of a prominent expression of the enzyme in brain, raise questions regarding its potential involvement in neurodegenerative disorders.
Assuntos
Apoptose , Caspase 6/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Caspase 6/genética , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Células HeLa , Humanos , Domínios Proteicos , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
SMSr/SAMD8 is an ER-resident ceramide phosphoethanolamine synthase with a critical role in controlling ER ceramides and suppressing ceramide-induced apoptosis in cultured cells. SMSr-mediated ceramide homeostasis relies on the enzyme's catalytic activity as well as on its N-terminal sterile α-motif or SAM domain. Here we report that SMSr-SAM is structurally and functionally related to the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. Native gel electrophoresis indicates that both SAM domains form homotypic oligomers. Chemical crosslinking studies show that SMSr self-associates into ER-resident trimers and hexamers that resemble the helical oligomers formed by DGKδ-SAM. Residues critical for DGKδ-SAM oligomerization are conserved in SMSr-SAM and their substitution causes a dissociation of SMSr oligomers as well as a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and Ca2+ homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Our data provide first demonstration of a multi-pass membrane protein that undergoes homotypic oligomerization via its SAM domain and indicate that SAM-mediated self-assembly of SMSr is required for efficient retention of the enzyme in the ER.
RESUMO
The fate of exogenous short-chain analogues of phosphatidylethanolamine and phosphatidylserine was studied in a deep-rough derivative of E. coli mutant strain AD93 that cannot synthesize phosphatidylethanolamine de novo. Using mass spectrometry, it was shown that dicaproyl(di 6:0)-phosphatidylethanolamine is extensively remodeled, eventually adopting the phosphatidylethanolamine species profile of the parental wild-type strain of AD93. Dicaproyl-phosphatidylserine was decarboxylated to form phosphatidylethanolamine, and yielded a species profile, which strongly resembled that of the introduced phosphatidylethanolamine. This demonstrates transport of phosphatidylserine to the cytosolic leaflet of the inner membrane. The changes of the species profile of phosphatidylethanolamine indicate that the short-chain phospholipids are most likely remodeled via two consecutive acyl chain substitutions, and at least part of this remodeling involves transport to the inner membrane.
Assuntos
Escherichia coli/metabolismo , Fosfatidiletanolaminas/metabolismo , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Espectrometria de MassasRESUMO
Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.
Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Azóis/farmacologia , Cromatografia em Camada Fina , Temperatura Baixa , Corantes Fluorescentes/farmacologia , Nitrobenzenos/farmacologia , Peptidoglicano Glicosiltransferase/metabolismo , Coloração e Rotulagem , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
Phospholipids are synthesized in biogenic membranes, but only on one leaflet of the bilayer. To support balanced growth of the membrane, phospholipid translocation, or flip-flop, has to occur. Though consensus has been reached that flip-flop is most likely mediated by (a) membrane-associated protein(s), a dedicated flippase has not been identified yet in any biogenic membrane. The characteristics of the flip-flop process are summarized, and possible mechanisms, including the need for a dedicated flippase, are discussed.
Assuntos
Bactérias/metabolismo , Proteínas de Transporte/fisiologia , Membrana Celular/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Transferência de Fosfolipídeos , Fosfolipídeos/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Escherichia coli/metabolismo , Modelos Biológicos , Peptídeos/química , Fatores de TempoRESUMO
Biogenic membranes contain the enzymes that synthesize the cell's membrane lipids, of which the phospholipids are the most widespread throughout nature. Being synthesized at and inserted into the cytoplasmic leaflet of biogenic membranes, the phospholipids must migrate to the opposite leaflet to ensure balanced growth of the membrane. In this review, the current knowledge of transbilayer movement of phospholipids in biogenic membranes is summarized and the available data are compared to what is known about lipid translocation in other membranes. On the basis of this, a mechanism is proposed, in which phospholipid translocation in biogenic membranes is mediated via membrane-spanning segments of a subset of proteins, characterized by a small number of transmembrane helices. We speculate that proteins of this subset facilitate lipid translocation via the protein-lipid interface, because they display more dynamic behavior and engage in less stable protein-lipid interactions than larger membrane proteins.
Assuntos
Bicamadas Lipídicas/química , Microdomínios da Membrana/química , Fosfolipídeos/química , Animais , Transporte Biológico , Humanos , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Fosfolipídeos/metabolismoRESUMO
The mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane alpha-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2-6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (C6-NBD)-phospholipids (Kol, M. A., de Kroon, A. I., Rijkers, D. T., Killian, J. A., and de Kruijff, B. (2001) Biochemistry 40, 10500-10506). Here the dithionite reduction assay was used to measure transbilayer equilibration of C6-NBD-phospholipids in proteoliposomes, composed of Escherichia coli phospholipids and a subset of bacterial membrane proteins. It is shown that two well characterized integral proteins of the bacterial cytoplasmic membrane, leader peptidase and the potassium channel KcsA, induce phospholipid translocation, most likely by their transmembrane domains. In contrast, the ATP-binding cassette transporter from the E. coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein from E. coli, did not facilitate flop either, demonstrating specificity of protein-mediated phospholipid translocation. The results are discussed in the light of phospholipid transport across the E. coli inner membrane.
Assuntos
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop). It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive. Previously, we have demonstrated flop of 2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl] (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E. coli [Kol et al. (2001) Biochemistry 40, 10500]. Here, we investigated whether the specific phospholipid composition of E. coli is a prerequisite for transmembrane helix-induced flop of phospholipids. This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay. The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures. The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC. Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop. Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue. Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.