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ABSTRACT: The pan-eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild-type and Arv1-deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport-coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild-type and arv1∆ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1∆ cells, specific defects affecting single C-terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail-anchored proteins involved in membrane homoeostasis.
Traffic 05/2013; · 4.92 Impact Factor
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ABSTRACT: Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Mazière, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101-115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t(½) < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)hexanoyl)-D-glucosyl-β1-1'-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer flippases. Direct tests showed that these proteins have no flippase activity.
Journal of Biological Chemistry 03/2012; 287(19):15523-32. · 4.77 Impact Factor
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ABSTRACT: Oxysterol binding protein-related proteins, including the yeast proteins encoded by the OSH gene family (OSH1-OSH7), are implicated in the non-vesicular transfer of sterols between intracellular membranes and the plasma membrane. In light of recent studies, we revisited the proposal that Osh proteins are sterol transfer proteins and present new models consistent with known Osh protein functions. These models focus on the role of Osh proteins as sterol-dependent regulators of phosphoinositide and sphingolipid pathways. In contrast to their posited role as non-vesicular sterol transfer proteins, we propose that Osh proteins coordinate lipid signaling and membrane reorganization with the assembly of tethering complexes to promote molecular exchanges at membrane contact sites.
Journal of Biological Chemistry 02/2012; 287(14):11481-8. · 4.77 Impact Factor
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ABSTRACT: Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol-binding protein (Osh1-7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔ osh4-1(ts)) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well as by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non-vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.
Traffic 06/2011; 12(10):1341-55. · 4.92 Impact Factor
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ABSTRACT: Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ<10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.
Current biology: CB 01/2011; 21(2):149-53. · 10.99 Impact Factor
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ABSTRACT: Mannose-phosphate-dolichol (MPD) is a multifunctional glycolipid that is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and used on the opposite side of the membrane in the ER lumen as a mannose donor for protein N-glycosylation, glycosylphosphatidylinositol-anchoring, and C- and O-mannosylation. For this, it must be translocated, i.e., flipped, across the ER membrane. The molecular identity of the MPD translocator (MPD flippase) is not known. Here we show that MPD-flippase activity can be reconstituted in large unilamellar proteoliposomes prepared from phosphatidylcholine and Triton X-100-solubilized rat liver ER-membrane proteins. Using carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl NO(+) as a topological probe to selectively oxidize MPD molecules in the outer leaflet of the reconstituted vesicles, we demonstrate rapid, protein-dependent, ATP-independent transbilayer translocation of MPD from the inner to the outer leaflet. MPD flipping is highly specific. A stereoisomer of MPD was weakly translocated (> 10-fold lower rate) compared with natural MPD. Competition experiments with water-soluble isoprenyl monophosphates showed that MPD flippase recognizes the dolichol chain of MPD, preferring a saturated alpha-isoprene to unsaturated trans- or cis- alpha-isoprene units. Chromatography of the detergent-solubilized ER protein mixture prior to reconstitution indicated that MPD flippase (i) is not a Con A-binding glycoprotein and (ii) can be resolved from the oligosaccharide-diphosphate dolichol flippase that translocates Man(5)GlcNAc(2)-PP-dolichol, a lipid intermediate of N-glycosylation. These data provide a mechanistic framework for understanding MPD flipping, as well as a biochemical basis for identifying MPD flippase.
Proceedings of the National Academy of Sciences 06/2010; 107(25):11289-94. · 9.68 Impact Factor
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ABSTRACT: The biosynthesis of glycoconjugates such as N-glycoproteins and GPI-anchored proteins in eukaryotes and cell wall peptidoglycan and lipopolysaccharide in bacteria requires lipid intermediates to be flipped rapidly across the endoplasmic reticulum or bacterial cytoplasmic membrane (so-called biogenic membranes). Rapid flipping is also required to normalize the number of glycerophospholipids in the two leaflets of the bilayer as the membrane expands in a growing cell. Although lipids diffuse rapidly in the plane of the membrane, the intrinsic rate at which they flip across membranes is very low. Biogenic membranes possess dedicated lipid transporters or flippases to increase flipping to a physiologically sufficient rate. The flippases are “ATP-independent” and facilitate “downhill” transport. Most predicted biogenic membrane flippases have not been identified at the molecular level, and the few flippases that have been identified by genetic approaches have not been biochemically validated. Here we summarize recent progress on this fundamental topic and speculate on the mechanism(s) by which biogenic membrane flippases facilitate transbilayer lipid movement.Keywords: Biogenic membrane: A “self-synthesizing” membrane, capable of synthesizing and integrating its lipid and protein components. Examples are the endoplasmic reticulum and the bacterial cytoplasmic membrane.; Flippase: Used here to signify a membrane protein that facilitates bidirectional, ATP-independent flipping of polar lipids across a biogenic membrane.; Flipping: Reorientation of a lipid from one leaflet of a membrane bilayer to the other. Used interchangeably with “flip-flop” and “transbilayer translocation”. Flip refers to transport of a lipid from the exoplasmic membrane leaflet to the cytoplasmic leaflet, i.e., out → in, whereas flop designates in → out transport.; Membrane protein: A protein that is integral to the membrane. A polytopic membrane protein spans the membrane many times.; Polar lipid: An amphipathic molecule, with a hydrophobic unit derived from fatty acids, sphingosine or isoprenoids (such as dolichol and undecaprenol), and a zwitterionic or charged hydrophilic headgroup. Typical examples are phosphatidylcholine, sphingomyelin and dolichyl phosphate mannose.
11/2009;
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ABSTRACT: The biosynthesis of glycoconjugates such as N-glycoproteins and GPI-anchored proteins in eukaryotes and cell wall peptidoglycan and lipopolysaccharide in bacteria requires lipid intermediates to be flipped rapidly across the endoplasmic reticulum or bacterial cytoplasmic membrane (so-called biogenic membranes). Rapid flipping is also required to normalize the number of glycerophospholipids in the two leaflets of the bilayer as the membrane expands in a growing cell. Although lipids diffuse rapidly in the plane of the membrane, the intrinsic rate at which they flip across membranes is very low. Biogenic membranes possess dedicated lipid transporters or flippases to increase flipping to a physiologically sufficient rate. The flippases are "ATP-independent" and facilitate "downhill" transport. Most predicted biogenic membrane flippases have not been identified at the molecular level, and the few flippases that have been identified by genetic approaches have not been biochemically validated. Here we summarize recent progress on this fundamental topic and speculate on the mechanism(s) by which biogenic membrane flippases facilitate transbilayer lipid movement.
ACS Chemical Biology 09/2009; 4(11):895-909. · 6.45 Impact Factor
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ABSTRACT: Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [(3)H]cholesterol in the form of [(3)H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [(3)H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [(3)H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Delta cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport.
Eukaryotic Cell 01/2009; 8(2):161-9. · 3.60 Impact Factor
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ABSTRACT: The oligosaccharide donor for protein N-glycosylation, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, is synthesized via a multistep pathway that starts on the cytoplasmic face of the endoplasmic reticulum (ER) and ends in the lumen where the glycosylation reaction occurs. This necessitates transbilayer translocation or flipping of the lipid intermediate Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) across the ER membrane. The mechanism by which M5-DLO-or any other lipid-is flipped across the ER is unknown, except that specific transport proteins or flippases are required. We recently demonstrated M5-DLO flipping activity in proteoliposomes reconstituted from detergent-solubilized ER membrane proteins and showed that it was ATP-independent and required a trypsin-sensitive protein that sedimented at approximately 4S. By using an activity-enriched fraction devoid of glycerophospholipid flippase activity, we now report that M5-DLO is rapidly flipped in the reconstituted system with a time constant tau <2 min, whereas its triantennary structural isomer is flipped slowly with tau >200 min. DLOs larger than M5-DLO are also poorly translocated, with tau ranging from approximately 10 min to >200 min. We conclude that (i) the number and arrangement of mannoses in the DLO glycan has a profound effect on the ability of the DLO to be translocated by the flippase, (ii) glycan size per se does not dictate whether a DLO will be flipped, and (iii) the flippase is highly specific for M5-DLO. Our results suggest a simple structural model for the interaction between the DLO head group and the flippase.
Proceedings of the National Academy of Sciences 01/2009; 106(3):767-72. · 9.68 Impact Factor
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ABSTRACT: Protein N-glycosylation requires flipping of the glycolipid Man(5)GlcNAc(2)-diphosphate dolichol (Man(5)GlcNAc(2)-PP-Dol) across the endoplasmic reticulum (ER). Helenius et al. report genetic evidence suggesting that Rft1, an essential ER membrane protein in yeast, is required directly to translocate Man(5)GlcNAc(2)-PP-Dol. We now show that a specific ER protein(s), but not Rft1, is required to flip Man(5)GlcNAc(2)-PP-Dol in reconstituted vesicles. Rft1 may have a critical accessory role in translocating Man(5)GlcNAc(2)-PP-Dol in vivo, but the Man(5)GlcNAc(2)-PP-Dol flippase itself remains to be identified.
Nature 08/2008; 454(7204):E3-4; discussion E4-5. · 36.28 Impact Factor
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Anant K Menon
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ABSTRACT: The Rho GTPase Cdc42 is essential for polarized growth of budding yeast. Temporal control of Cdc42 depends partly on the activity of its GTPase-activating proteins (GAPs). In this issue of Developmental Cell, Saito et al. report that Cdc42 GAP activity is regulated by the phospholipid composition of the bud-tip membrane, under control of the phospholipid flippases Lem3-Dnf1 and Lem3-Dnf2.
Developmental Cell 12/2007; 13(5):607-8. · 14.03 Impact Factor
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ABSTRACT: Short cationic oligomers, including arginine-rich peptides and analogous beta-amino acid oligomers ("beta-peptides"), can enter the cytoplasm and nucleus of a living cell from the extracellular medium. It seems increasingly clear that multiple entry pathways are possible, depending upon the structure of the guanidinium-rich molecule, the type of cell, and other factors. We have previously shown that conformational stability and spatial clustering of guanidinium groups increase the HeLa cell entry efficiency of short helical beta-peptides bearing six guanidinium groups, results that suggest that these beta-peptides could be useful tools for studying the entry process. Here we describe studies intended to identify the point in the entry process at which helix stability and spatial arrangement of guanidinium groups exert their effect. Our results suggest that key distinctions involve the mode of interaction between different guanidinium-rich beta-peptides and the HeLa cell surface. A specific guanidinium display appears to be required for proper engagement of cell-surface heparan sulfate proteoglycans and concomitant induction of endocytic uptake.
ChemBioChem 06/2007; 8(8):917-26. · 3.94 Impact Factor
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ABSTRACT: Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring >20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane. The completed GPI anchor is attached to proteins that have been translocated across the ER membrane and that display a GPI signal anchor sequence at the C terminus. GPI proteins transit the secretory pathway to the cell surface; in yeast, many become covalently attached to the cell wall. Genes encoding proteins involved in all but one of the predicted steps in the assembly of the GPI precursor glycolipid and its transfer to protein in mammals and yeast have now been identified. Most of these genes encode polytopic membrane proteins, some of which are organized in complexes. The steps in GPI assembly, and the enzymes that carry them out, are highly conserved. GPI biosynthesis is essential for viability in yeast and for embryonic development in mammals. In this review, we describe the biosynthesis of mammalian and yeast GPIs, their transfer to protein, and their subsequent processing.
The Journal of Lipid Research 05/2007; 48(5):993-1011. · 5.56 Impact Factor
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ABSTRACT: De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.
Eukaryotic Cell 03/2007; 6(3):454-64. · 3.60 Impact Factor
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ABSTRACT: Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.
Journal of Biological Chemistry 01/2007; 281(50):38358-64. · 4.77 Impact Factor
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ABSTRACT: Sterols are important components of many biological membranes, and changes in sterol levels can have dramatic effects on membrane properties. Sterols are transported rapidly between cellular organelles by vesicular and nonvesicular processes. Recent studies have identified transmembrane proteins that facilitate the removal of sterols from membranes as well as soluble cytoplasmic proteins that play a role in their movement through the cytoplasm. The mechanisms by which these proteins work are generally not well understood. Cells maintain large differences in the sterol:phospholipid ratio in different organelles. Recent theoretical and experimental studies indicate ways in which the lipid environment can alter the chemical potential of sterols, which may help to explain aspects of their transport kinetics and distribution.
Current Opinion in Cell Biology 09/2006; 18(4):379-85. · 12.90 Impact Factor
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Amy E Gruszynski,
Frederick J van Deursen,
Maria C Albareda,
Alexander Best,
Kshitiz Chaudhary,
Laura J Cliffe,
Laura del Rio,
Joe Dan Dunn,
Louise Ellis,
Krystal J Evans, [......],
Nicholas A Malmquist,
Yusuf Omosun,
Jennifer B Palenchar,
Sara Prickett,
George A Punkosdy,
Giel van Dooren,
Qian Wang, Anant K Menon,
Keith R Matthews,
James D Bangs
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ABSTRACT: African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.
Molecular and Biochemical Parasitology 07/2006; 147(2):211-23. · 2.55 Impact Factor
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ABSTRACT: After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1(282), a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented, N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1(282) had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1(282) appended to alpha2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1(282), and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins.
Journal of Biological Chemistry 05/2005; 280(16):16402-9. · 4.77 Impact Factor
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ABSTRACT: The mechanism by which newly synthesized sterols are transported from their site of synthesis, the endoplasmic reticulum (ER), to the sterol-enriched plasma membrane (PM) is not fully understood. Studies in mammalian cells suggest that newly synthesized cholesterol is transported to the PM in Golgi-bypassing vesicles and/or via a nonvesicular process. Using the yeast Saccharomyces cerevisiae as a model system, we now rule out an essential role for known vesicular transport pathways in transporting the major yeast sterol, ergosterol, from its site of synthesis to the PM. We use a cyclodextrin-based sterol capture assay to show that transport of newly synthesized ergosterol to the PM is unaltered in cells defective in Sec18p, a protein required for almost all intracellular vesicular trafficking events; we also show that transport is not blocked in cells that are defective in formation of transport vesicles at the ER or in vesicle fusion with the PM. Our data suggest instead that transport occurs by equilibration (t(1/2) approximately 10-15 min) of ER and PM ergosterol pools via a bidirectional, nonvesicular process that is saturated in wild-type exponentially growing yeast. To reconcile an equilibration process with the high ergosterol concentration of the PM relative to ER, we note that a large fraction of PM ergosterol is found condensed with sphingolipids in membrane rafts that coexist with free sterol. We propose that the concentration of free sterol is similar in the PM and ER and that only free (nonraft) sterol molecules have access to a nonvesicular transport pathway that connects the two organelles. This is the first description of biosynthetic sterol transport in yeast.
Biochemistry 05/2005; 44(15):5816-26. · 3.42 Impact Factor
