RESUMO
The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multistep pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 congenital disorders of glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multispanning membrane protein located in the ER, with its N and C termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.
RESUMO
Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.
Assuntos
Fosfolipídeos , Receptores Acoplados a Proteínas G , Animais , Transporte Biológico , Colesterol , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Bovinos , PerusRESUMO
SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Glicosilfosfatidilinositóis , Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Lipogênese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
Opsins, the protein moieties of animal visual photo-pigments, have emerged as moonlighting proteins with diverse, light-dependent and -independent physiological functions. This raises the need to revise some basic assumptions concerning opsin expression, structure, classification, and evolution.
Assuntos
Evolução Molecular , Opsinas , Animais , Opsinas/genética , Opsinas/metabolismo , Filogenia , Pigmentos da Retina , Opsinas de Bastonetes/genéticaRESUMO
Glycosylphosphatidylinositol (GPI)-anchored proteins are found in all eukaryotes and are especially abundant on the surface of protozoan parasites such as Trypanosoma brucei. GPI-mannosyltransferase-I (GPI-MT-I) catalyzes the addition of the first of three mannoses that make up the glycan core of GPI. Mammalian and yeast GPI-MT-I consist of two essential subunits, the catalytic subunit PIG-M/Gpi14 and the accessory subunit PIG-X/Pbn1(mammals/yeast). T. brucei GPI-MT-I has been highlighted as a potential antitrypanosome drug target but has not been fully characterized. Here, we show that T. brucei GPI-MT-I also has two subunits, TbGPI14 and TbPBN1. Using TbGPI14 deletion, and TbPBN1 RNAi-mediated depletion, we show that both proteins are essential for the mannosyltransferase activity needed for GPI synthesis and surface expression of GPI-anchored proteins. In addition, using native PAGE and co-immunoprecipitation analyses, we demonstrate that TbGPI14 and TbPBN1 interact to form a higher-order complex. Finally, we show that yeast Gpi14 does not restore GPI-MT-I function in TbGPI14 knockout trypanosomes, consistent with previously demonstrated species specificity within GPI-MT-I subunit associations. The identification of an essential trypanosome GPI-MT-I subcomponent indicates wide conservation of the heterodimeric architecture unusual for a glycosyltransferase, leaving open the question of the role of the noncatalytic TbPBN1 subunit in GPI-MT-I function.
Assuntos
Trypanosoma brucei brucei , Animais , Glicosilfosfatidilinositóis , Mamíferos/metabolismo , Manosiltransferases/genética , Manosiltransferases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Many eukaryotic cell-surface proteins are post-translationally modified by a glycosylphosphatidylinositol (GPI) moiety that anchors them to the cell membrane. The biosynthesis of GPI anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol. This reaction is catalyzed by GPI GlcNAc transferase, a multisubunit complex comprising the catalytic subunit Gpi3/PIG-A as well as at least five other subunits, including the hydrophobic protein Gpi2, which is essential for the activity of the complex in yeast and mammals, but the function of which is not known. To investigate the role of Gpi2, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism that initially provided the first insights into GPI structure and biosynthesis. We generated insect-stage (procyclic) trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites, (i) GPI GlcNAc transferase activity is reduced, but not lost, in contrast with yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of procyclins, the major surface proteins, are underglycosylated when compared with their WT counterparts, indicating the importance of TbGPI2 for reactions that occur in the Golgi apparatus. Immunofluorescence microscopy localized TbGPI2 not only to the endoplasmic reticulum but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic noncanonical role in Golgi-localized GPI anchor modification in trypanosomes.
Assuntos
Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Complexo de Golgi/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Polissacarídeos/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanossomíase/metabolismo , Animais , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/química , Proteínas de Protozoários , Trypanosoma brucei brucei/isolamento & purificação , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase/parasitologia , Tripanossomíase/patologiaRESUMO
Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by nonvesicular mechanisms requiring sterol transport proteins (STPs). Here we examine the idea that transport is enhanced at membrane contact sites where the ER is closely apposed to the PM. We conclude that sterol desorption from the membrane, rather than STP-mediated diffusion, is rate limiting in the cellular context, so there is no apparent kinetic benefit to having STP-mediated sterol transfer occur at contact sites. Contact sites may instead compartmentalize lipid synthesis or transport machinery, providing opportunities for regulation.
Assuntos
Esteróis/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , HumanosRESUMO
Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.
Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fosfolipídeos/metabolismo , Esteróis/metabolismo , Lipídeos/biossíntese , Proteínas de Membrana/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , LevedurasRESUMO
Phospholipid scramblases externalize phosphatidylserine to facilitate numerous physiological processes. Several members of the structurally unrelated TMEM16 and G protein-coupled receptor (GPCR) protein families mediate phospholipid scrambling. The structure of a TMEM16 scramblase shows a membrane-exposed hydrophilic cavity, suggesting that scrambling occurs via the ?credit-card" mechanism where lipid headgroups permeate through the cavity while their tails remain associated with the membrane core. Here we show that afTMEM16 and opsin, representatives of the TMEM16 and GCPR scramblase families, transport phospholipids with polyethylene glycol headgroups whose globular dimensions are much larger than the width of the cavity. This suggests that transport of these large headgroups occurs outside rather than within the cavity. These large lipids are scrambled at rates comparable to those of normal phospholipids and their presence in the reconstituted vesicles promotes scrambling of normal phospholipids. This suggests that both large and small phospholipids can move outside the cavity. We propose that the conformational rearrangements underlying TMEM16- and GPCR-mediated credit-card scrambling locally deform the membrane to allow transbilayer lipid translocation outside the cavity and that both mechanisms underlie transport of normal phospholipids.
Assuntos
Anoctaminas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Anoctaminas/genética , Transporte Biológico Ativo/fisiologia , Células HEK293 , Humanos , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Acoplados a Proteínas G/genética , Saccharomyces cerevisiaeRESUMO
Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.
Assuntos
Membrana Celular/metabolismo , Ergosterol/metabolismo , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Membrana Celular/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismoRESUMO
Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins. Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional-as yet unidentified-sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs. We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/genética , Citosol/metabolismo , Ergosterol/metabolismo , Transporte Proteico , Proteômica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The lipid composition of cellular organelles is tailored to suit their specialized tasks. A fundamental transition in the lipid landscape divides the secretory pathway in early and late membrane territories, allowing an adaptation from biogenic to barrier functions. Defending the contrasting features of these territories against erosion by vesicular traffic poses a major logistical problem. To this end, cells evolved a network of lipid composition sensors and pipelines along which lipids are moved by non-vesicular mechanisms. We review recent insights into the molecular basis of this regulatory network and consider examples in which malfunction of its components leads to system failure and disease.
Assuntos
Homeostase , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Animais , Proteínas de Transporte/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/análise , Lipídeos de Membrana/biossíntese , Organelas/química , Organelas/metabolismo , Esfingolipídeos/metabolismoRESUMO
The chemical synthesis of molecular probes to identify and study membrane proteins involved in the biological pathway of protein glycosylation is described. Two short-chain glycolipid analogs that mimic the naturally occurring substrate mannosyl phosphoryl dolichol exhibit either photoreactive and clickable properties or allow the use of a fluorescence readout. Both probes consist of a hydrophilic mannose headgroup that is linked to a citronellol derivative via a phosphodiester bridge. Moreover, a novel phosphoramidite chemistry-based method offers a straightforward approach for the non-enzymatic incorporation of the saccharide moiety in an anomerically pure form.
RESUMO
Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca2+-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus Nectria hematococca) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides the transbilayer conduit for lipids, but the mechanism by which Ca2+ regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity, we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca2+-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the nhTMEM16 homodimer and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing an acyl carrier protein (ACP) tag at one or both of the termini. These constructs were fluorescently labeled by ACP synthase-mediated insertion of CoA-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer. We now report that the measured distances do not vary significantly between Ca2+-replete and EGTA-treated samples, indicating that whereas the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change.
Assuntos
Anoctaminas/química , Anoctaminas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Bicamadas Lipídicas/metabolismo , Nectria/enzimologia , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/genética , Transporte Biológico , Cálcio/metabolismo , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Dimerização , Fluorescência , Proteínas Fúngicas/genética , Bicamadas Lipídicas/química , Nectria/química , Nectria/genética , Proteínas de Transferência de Fosfolipídeos/genéticaRESUMO
Members of the G protein-coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([3H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [3H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [3H]PI hydrolysis increased, indicating that [3H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC-based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase.
Assuntos
Anoctaminas/metabolismo , Proteínas Fúngicas/metabolismo , Opsinas/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Anoctaminas/química , Anoctaminas/genética , Transporte Biológico , Bovinos , Fluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hidrólise , Cinética , Nectria/enzimologia , Opsinas/química , Opsinas/genética , Fosfatidilinositóis/química , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein-related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1-Lam6, in the yeast Saccharomyces cerevisiae. Lam1-Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of â¼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling "head-first" entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress.
Assuntos
Antiporters/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Esteróis/química , Antiporters/genética , Antiporters/metabolismo , Transporte Biológico Ativo/fisiologia , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esteróis/metabolismoRESUMO
The membrane protein RFT1 is essential for normal protein N-glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid Man5GlcNAc2-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background. We now report that lack of T. brucei RFT1 (TbRFT1) not only affects protein N-glycosylation but also glycosylphosphatidylinositol (GPI) anchor side-chain modification. Analysis by immunoblotting, metabolic labeling, and mass spectrometry demonstrated that the major GPI-anchored proteins of T. brucei procyclic forms have truncated GPI anchor side chains in TbRFT1 null parasites when compared with wild-type cells, a defect that is corrected by expressing a tagged copy of TbRFT1 in the null background. In vivo and in vitro labeling experiments using radiolabeled GPI precursors showed that GPI underglycosylation was not the result of decreased formation of the GPI precursor lipid or defective galactosylation of GPI intermediates in the endoplasmic reticulum, but rather due to modifications that are expected to occur in the Golgi apparatus. Unexpectedly, immunofluorescence microscopy localized TbRFT1 to both the endoplasmic reticulum and the Golgi, consistent with the proposal that TbRFT1 plays a direct or indirect role in GPI anchor glycosylation in the Golgi apparatus. Our results implicate RFT1 in a wider range of glycosylation processes than previously appreciated.
Assuntos
Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicosilação , Complexo de Golgi/genética , Glicoproteínas de Membrana/genética , Oligossacarídeos/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genéticaRESUMO
BACKGROUND: The lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. While the assay has yielded insight into the scramblase activity in crude membrane preparations, functional validation of candidate scramblases, stoichiometry of scramblase complexes as well as ATP-dependence of flippases, data analysis in its context has remained a task involving many manual steps. RESULTS: With the extension package "flippant" to R, a free software environment for statistical computing and graphics, we introduce an integrated solution for the analysis and publication-grade graphical presentation of dithionite scramblase assays and demonstrate its utility in revisiting an originally manual analysis from the publication record, closely reproducing the reported results. CONCLUSIONS: "flippant" allows for quick, reproducible data analysis of scramblase activity assays and provides a platform for review, dissemination and extension of the strategies it employs.
Assuntos
Bioquímica/métodos , Lipídeos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Software , Fluorescência , Humanos , Proteínas de Transferência de Fosfolipídeos/análiseRESUMO
Glycerophospholipids are the principal fabric of cellular membranes. The pathways by which these lipids are synthesized were elucidated mainly through the work of Kennedy and colleagues in the late 1950s and early 1960s. Subsequently, attention turned to cell biological aspects of lipids: Where in the cell are lipids synthesized? How are lipids integrated into membranes to form a bilayer? How are they sorted and transported from their site of synthesis to other cellular destinations? These topics, collectively termed 'lipid topogenesis', were the subject of a review article in 1981 by Bell, Ballas and Coleman. We now assess what has been learned about early events of lipid topogenesis, i.e. "lipid synthesis, the integration of lipids into membranes, and lipid translocation across membranes", in the 35 years since the publication of this important review. We highlight the recent elucidation of the X-ray structures of key membrane enzymes of glycerophospholipid synthesis, progress on identifying lipid scramblase proteins needed to equilibrate lipids across membranes, and new complexities in the subcellular location and membrane topology of phosphatidylinositol synthesis revealed through a comparison of two unicellular model eukaryotes. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.
Assuntos
Membrana Celular/metabolismo , Glicerofosfolipídeos/biossíntese , Animais , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/metabolismo , Membrana Celular/química , Diglicerídeos de Citidina Difosfato/biossíntese , Glicerofosfolipídeos/química , Humanos , Bicamadas Lipídicas/metabolismo , Redes e Vias Metabólicas/fisiologia , Mitocôndrias/metabolismoRESUMO
The TMEM16 family comprises Ca2+-activated Cl- channels and phospholipid scramblases. The crystal structure of a fungal homolog, nhTMEM16, revealed an important architectural feature of this protein family in the form of a bilayer-spanning hydrophilic groove that is directly exposed to the membrane. This groove likely provides a pathway for lipid translocation. As mutations that alter ion channel activity of the TMEM16 proteins localize around the groove, it was suggested that the ion and lipid pathways coincide such that the ion pore is partly lined by phospholipids. However, this proposal was not supported by the observation that nhTMEM16 does not mediate ion transport. Here we show that nhTMEM16 mediates both ion and lipid transport and that its properties closely resemble those of a previously characterized fungal homolog, afTMEM16. We show that the reported lack of ion transport activity of nhTMEM16 is due to the lipid composition of the reconstitution membranes and to the presence of a GFP tag. Thus, nhTMEM16, like afTMEM16 and the mammalian TMEM16F, mediates simultaneous lipid scrambling and nonspecific ion transport. This supports the hypothesis that these two processes are tightly correlated and likely to be a general functional feature of the TMEM16 scramblases and therefore of general importance in understanding their biological roles.