MEMBRANE BIOGENESIS:LIPID SYNTHESIS AND INTRACELLULAR TRANSPORT

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	4050	1000	50%
305	4050	1030	50%

Progress 10/01/93 to 09/30/05

Outputs
We are interested in aspects of cellular membrane biogenesis concerned with lipid synthesis and intracellular transport. Our research is focused on assembly of glycosyl-phosphatidylinositols - GPIs - and GPI-anchored proteins, phospholipid flip-flop in biogenic membranes, sterol transport from the ER to the plasma membrane, and peptide translocation across membranes. GPI BIOSYNTHESIS: GPIs exist in all eukaryotic cells. GPIs are synthesized via a topologically complex pathway in the endoplasmic reticulum (ER). We identified localization motifs that retain the Gaa1 and PIG-T subunits of the GPI anchoring enzyme in the ER. We also showed that PIG-L, the second enzyme of GPI biosynthesis, forms a stoichiometric complex with PIG-H, a component of the multi-subunit glycosyltransferase responsible for initiating GPI biosynthesis. In a new effort, we used fluorescently labeled GPIs to show for the first time that early GPI intermediates are able to flip-flop in a protein-dependent, ATP-independent fashion across the ER membrane. Such flip-flop is postulated in current models of GPI assembly. PHOSPHOLIPID FLIPPASES: Glycerophospholipids are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and must be flipped across the ER to allow uniform bilayer expansion. Specific proteins, flippases, are needed to facilitate phospholipid flip-flop. We initiated efforts to purify the phospholipid flippase from detergent extracts of rat liver endoplasmic reticulum. We identified three key chromatographic steps using immobilized lectins and dye resins that permit a significant enrichment of flippase activity. Also, in a collaborative effort, we synthesized a complete set of fluorescence-labeled stereoisomers of phosphatidylinositol and used these to show that the ER flippase activity is indifferent to the stereochemistry of the phospholipid. This is an important result with consequences for models of cellular evolution. STEROL TRANSPORT: The mechanisms involved in the transport of sterols from their site of synthesis to the plasma membrane are unknown. Sterols are synthesized in the ER and transported to the sterol-rich plasma membrane (PM). Using yeast, we showed that ER-PM sterol transport occurs via a bidirectional equilibration process that does not require the classical secretory pathway. We tested a model for sterol homeostasis based on the assumption that a large fraction of PM sterol exists in the form of sterol-sphingolipid-enriched rafts. To identify the basis of non-vesicular sterol transport, we initiated studies to test candidate transport proteins. We also developed a selection scheme to enrich for yeast mutants defective in sterol transport. PEPTIDE TRANSLOCATION: We are studying the transmembrane transport of highly charged peptides such as the HIV Tat peptide. We used our ability to control beta-peptide secondary structure to explore the effects of conformational stability and geometry of guanidinium display on cell entry. We showed that both factors affect of beta-peptide accumulation in the cytoplasm and nucleus of HeLa cells without affecting cell surface binding.

Impacts
This work has fundamental impact on our understanding of membrane assembly processes in cells.

Publications

Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via nonvesicular equilibration.Biochemistry. 2005 Apr 19;44(15):5816-26.PMID: 15823040 [PubMed - indexed for MEDLINE]
Effects of conformational stability and geometry of guanidinium display on cell entry by beta-peptides.J Am Chem Soc. 2005 Mar 23;127(11):3686-7. No abstract available.PMID: 15771489 [PubMed - indexed for MEDLINE]
Endoplasmic reticulum localization of Gaa1 and PIG-T, subunits of the glycosylphosphatidylinositol transamidase complex.J Biol Chem. 2005 Apr 22;280(16):16402-9. Epub 2005 Feb 15.PMID: 15713669 [PubMed - indexed for MEDLINE]
Flip-flop of glycosylphosphatidylinositols (GPI's) across the ER. Chem Commun (Camb). 2005 Jan 28;(4):453-5. Epub 2004 Dec 2. PMID: 15654367 [PubMed - in process]

Progress 01/01/04 to 12/31/04

Outputs
We are interested in aspects of cellular membrane biogenesis concerned with lipid synthesis and intracellular transport. Our research is focused on assembly of glycosyl-phosphatidylinositols - GPIs - and GPI-anchored proteins, phospholipid flip-flop in biogenic membranes, sterol transport from the ER to the plasma membrane, and peptide translocation across membranes. GPI BIOSYNTHESIS: GPIs exist in all eukaryotic cells. GPIs are synthesized via a topologically complex pathway in the endoplasmic reticulum (ER). We identified localization motifs that retain the Gaa1 and PIG-T subunits of the GPI anchoring enzyme in the ER. We also showed that PIG-L, the second enzyme of GPI biosynthesis, forms a stoichiometric complex with PIG-H, a component of the multi-subunit glycosyltransferase responsible for initiating GPI biosynthesis. In a new effort, we used fluorescently labeled GPIs to show for the first time that early GPI intermediates are able to flip-flop in a protein-dependent, ATP-independent fashion across the ER membrane. Such flip-flop is postulated in current models of GPI assembly. PHOSPHOLIPID FLIPPASES: Glycerophospholipids are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and must be flipped across the ER to allow uniform bilayer expansion. Specific proteins, flippases, are needed to facilitate phospholipid flip-flop. We initiated efforts to purify the phospholipid flippase from detergent extracts of rat liver endoplasmic reticulum. We identified three key chromatographic steps using immobilized lectins and dye resins that permit a significant enrichment of flippase activity. Also, in a collaborative effort, we synthesized a complete set of fluorescence-labeled stereoisomers of phosphatidylinositol and used these to show that the ER flippase activity is indifferent to the stereochemistry of the phospholipid. This is an important result with consequences for models of cellular evolution. STEROL TRANSPORT: The mechanisms involved in the transport of sterols from their site of synthesis to the plasma membrane are unknown. Sterols are synthesized in the ER and transported to the sterol-rich plasma membrane (PM). Using yeast, we showed that ER-PM sterol transport occurs via a bidirectional equilibration process that does not require the classical secretory pathway. We tested a model for sterol homeostasis based on the assumption that a large fraction of PM sterol exists in the form of sterol-sphingolipid-enriched rafts. To identify the basis of non-vesicular sterol transport, we initiated studies to test candidate transport proteins. We also developed a selection scheme to enrich for yeast mutants defective in sterol transport. PEPTIDE TRANSLOCATION: We are studying the transmembrane transport of highly charged peptides such as the HIV Tat peptide. We used our ability to control beta-peptide secondary structure to explore the effects of conformational stability and geometry of guanidinium display on cell entry. We showed that both factors affect of beta-peptide accumulation in the cytoplasm and nucleus of HeLa cells without affecting cell surface binding.

Impacts
This work has fundamental impact on our understanding of membrane assembly processes in cells.

Publications

Pottekat, A. & Menon, A.K. (2004) Subcellular localization and targeting of PIG-L, the GlcNAc-PI-de-N-acetylase in the GPI biosynthetic pathway. J. Biol. Chem. 279, 15743-15751.
Chang, Q., Gummadi, S.N. & Menon, A.K. (2004) Chemical modification identifies two functionally distinct populations of glycerophospholipid flippases in rat liver ER. Biochemistry 43, 10710-10718.
Vishwakarma, R.A. and Menon, A.K. (2005) Flip-flop of glycosylphosphatidylinositols (GPI's) across the ER. Chem. Commun. 2005, 453-455.
Potocky, T., Menon, A.K., and Gellman, S.H. (2005) Effects of conformational stability and geometry of guanidium display on cell entry by b-peptides. J. Am. Chem. Soc. 127, 3686-3687.
Vainauskas, S. and Menon, A.K. (2005) Endoplasmic reticulum localization of Gaa1 and PIG-T, subunits of the glycosylphosphatidylinositol (GPI) transamidase complex. J. Biol. Chem. In press.
Vishwakarma, R.A., Vehring, S., Mehta, A., Sinha, A., Pomorski, T., Herrmann, A. and Menon, A.K. (2005) New fluorescent probes reveal that flippase-mediated flip-flop of phosphatidylinositol across the endoplasmic reticulum membrane does not depend on the stereochemistry of the lipid. Org. Biomol. Chem. DOI: 10.1039/B500300H.
Baumann, N.A., Sullivan, D.P., Ohvo-Rekila, H., Simonot, C., Pottekat, A., Klaassen, Z., Beh, C.T. & Menon, A.K. (2005) Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via non-vesicular equilibration. Biochemistry In press.

Progress 01/01/03 to 12/31/03

Outputs
We are interested in aspects of cellular membrane biogenesis concerned with lipid synthesis and intracellular transport. Our research is focused on assembly of glycosyl-phosphatidylinositols - GPIs - and GPI-anchored proteins, phospholipid flip-flop in biogenic membranes, sterol transport from the ER to the plasma membrane, and peptide translocation across membranes. GPI BIOSYNTHESIS: GPIs exist in all eukaryotic cells and serve to anchor a variety of proteins to cell membranes. GPIs are synthesized in the endoplasmic reticulum (ER). We identified localization motifs that retain the second enzyme of the pathway in the ER. Our work suggests that the motifs serve to multimerize the protein, thus blocking ER exit. We also studied the multi-subunit, membrane-bound enzyme (GPI transamidase) that is responsible for attaching GPI anchors to proteins. We analyzed Gaa1, a polytopic membrane protein subunit of GPI transamidase, and showed that a centrally located proline residue in its final transmembrane span is critical for GPI binding. PHOSPHOLIPID FLIPPASES: Glycerophospholipids are synthesized on the cytoplasmic face of biogenic membranes. The lipids must translocate across the bilayer to populate the exoplasmic leaflet for membrane assembly. We developed assays using fluorescent phospholipid analogs and a reconstitution strategy starting with rat liver ER membranes to progress towards purifying a phospholipid flippase from the ER. We also showed that there exist two distinct flippase populations, operationally defined by their differential sensitivity to protein modification reagents. STEROL TRANSPORT: The mechanisms involved in the transport of sterols from their site of synthesis to the plasma membrane are unknown. Sterols are synthesized in the ER and transported to the sterol-rich plasma membrane (PM). Using yeast, we showed that ER-PM sterol transport occurs via a bidirectional equilibration process. Transport proceeds normally in sec18-defective cells and is modulated, but not blocked, in other sec mutants. However, transport is slowed in sphingolipid-deficient lcb1-100 cells. Acknowledging that a large fraction of PM sterol exists in the form of sterol-sphingolipid-enriched rafts our results support a model in which raft formation lowers free sterol concentration in the PM to a level similar to that in the ER. We propose that ER-PM sterol traffic is a vesicle-independent partitioning process possibly mediated by numerous ER-PM membrane contact sites. The slower equilibration seen in lcb1-100 can be traced to an enlargement of the free sterol pool in the PM, stemming from the chronically lower sphingolipid content of the cells. PEPTIDE TRANSLOCATION: We are studying the transport of highly charged peptides (derived from the HIV TAT protein, as well as non-natural beta peptides) from the extracellular space to the cytoplasm and nucleus of living cells. Using fluorescent labeled peptides and confocal fluorescence microsocopy we show that the peptides are taken up into endocytic vesicles, then exit these vesicles to enter the cytoplasm. Endosomal exit of the peptides is regulated by endosome pH.

Impacts
This work has fundamental impact on our understanding of membrane assembly processes in cells.

Publications

Gummadi, S.N., Hrafnsdottir, S., Walent, J., Watkins, W.E. & Menon, A.K. (2003) Reconstitution and assay of biogenic membrane-derived phospholipid flippase activity in proteoliposomes. In Membrane Protein Protocols, B.S. Selinsky (ed.), Humana Press series `Methods in Molecular Biology', 228: 271-279.
Kostova, Z., Yan, B.C., Vainauskas, S., Schwartz, R., Menon, A.K. & Orlean, P. (2003) Comparative importance in vivo of conserved glutamates in the EX7E-motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in GPI assembly. Eur. J. Biochem. 270: 4507-4514.
Potocky, T., Menon, A.K. & Gellman, S.H. (2003) Cytoplasmic and nuclear delivery of a TAT-derived peptide and a b-peptide after endocytic uptake into HeLa cells. J. Biol. Chem. 278: 50188-50194.
Vainauskas, S. & Menon, A.K. (2003) Gaa1, a subunit of the GPI transamidase complex, is required for recognition of the GPI moiety. J. Biol. Chem. 278: In press.
Menon, A.K. (2003) Glycosylphosphatidylinositol (GPI) Anchors. In Encyclopedia of Biological Chemistry, W.J. Lennarz, M.D. Lane (eds.), Elsevier. In press.

Progress 01/01/02 to 12/31/02

Outputs
We are interested in aspects of cellular membrane biogenesis concerned with lipid synthesis and intracellular transport. Our research is focused on assembly of GPI-anchored proteins, phospholipid flip-flop in biogenic membranes, and sterol transport from the ER to the plasma membrane. GPI ANCHORING OF PROTEINS AND INTRACELLULAR TRAFFICKING OF GPIs : An abstract of our recent work is given here: Gaa1 is a subunit of the endoplasmic reticulum (ER)-localized GPI transamidase complex. Gaa1 has a cytoplasmically oriented N-terminal sequence with a potential membrane sorting signal in the form of an internal triple arginine motif, and a single N-glycosylated loop between the first and second transmembrane domains through which it interacts with other transamidase subunits. To determine the role of the N-terminal sequence in subcellular sorting, we analyzed Gaa1 variants transiently expressed in HeLa cells. Our data show that (i) Gaa1 constructs are endoproteolytically cleaved within the lumenal loop to yield a membrane-bound glycoprotein fragment (N-fragment) anchored by the first transmembrane segment, (ii) the N-fragment cannot interact with other transamidase subunits and is readily degraded, and (iii) N-fragments lacking the RRR motif are protected from degradation and accumulate in a post-ER compartment. Our analyses suggest that the Gaa1 N-terminal sequence contains determinants, embodied at least in part by the RRR motif, that act to retain the protein in the ER prior to its assembly into the transamidase complex and that also signal degradation of Gaa1 N-fragments. These data provide the first example of signal mediated retention and degradation of a membrane protein subunit of an ER-localized protein complex. STEROL TRANSPORT: The mechanisms involved in the transport of sterols from their site of synthesis to the plasma membrane are unknown. Sterols are synthesized in the endoplasmic reticulum (ER) and transported to the sterol-enriched plasma membrane (PM) against a bulk concentration gradient. Using yeast, we show that a pulse of [3H-methyl]ergosterol is transported to a methyl-b-cyclodextrin (MbCD)-accessible PM pool via an isotopic equilibration process (t1/2 10 min) that is blocked on ice. PM delivery of [3H]ergosterol occurs normally in sec18-defective cells and is modulated, but not blocked, in other sec mutants. Transport is slowed in lcb1-100 cells but is unaffected by myriocin, a drug that inhibits Lcb1p function. Acknowledging that a large fraction of PM sterol exists in the form of sterol-sphingolipid-enriched rafts our results support a model in which raft formation lowers free sterol concentration in the PM to a level similar to that in the ER. We propose that ER-PM sterol traffic is a vesicle-independent partitioning process possibly mediated by numerous ER-PM membrane contact sites. The slower equilibration seen in lcb1-100 can be traced to an enlargement of the free sterol pool in the PM, stemming from the chronically lower sphingolipid content of the cells.

Impacts
This work has fundamental impact on our understanding of membrane assembly processes in cells.

Publications

Kubelt, J., Menon, A.K., Muller, P., and Herrmann, A. (2002) Transbilayer movement of fluorescent phospholipid analogs in the cytoplasmic membrane of E. coli. Biochemistry, 41, 5605-5612.
Menon, A.K. (2002) Introduction: lipid transport - an overview (editorial). Seminars in Cell and Developmental Biology, 13, 159-162.
Gummadi, S.N., and Menon, A.K. (2002) Transbilayer movement of dipalmitoylphosphatidylcholine in proteoliposomes reconstituted from detergent extracts of endoplasmic reticulum: kinetics of transbilayer transport mediated by a single flippase and identification of protein fractions enriched in flippase activity. J. Biol. Chem., 277, 25337-25343.
Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A.K. (2002) Structural requirements for the recruitment of Gaa1 into a functional glycosylphosphatidylinositol transamidase complex. J. Biol. Chem., 277, 30535-30542.

Progress 01/01/01 to 12/31/01

Outputs
GPI ANCHORING OF PROTEINS AND INTRACELLULAR TRAFFICKING OF GPIs : GPIs represent a family of glycolipids that exist in all eukaryotic cells and serve to anchor a variety of proteins to cell membranes. Using photocrosslinking techniques, initial exploration of the protein biochemical environment encountered by a pro-protein during its conversion to a GPI-anchored protein have yielded the first direct evidence that genetically identified components of the anchoring machinery are in close proximity to the pro-protein, and indicated the presence of additional protein subunits in the anchoring machinery. Attempting to dissect the molecular architecture of the transamidase complex, we prepared a series of truncation mutants of the polytopic transamidase subunit Gaa1p - these mutants were expressed in cells, analyzed for their localization and membrane topology, and tested for their ability to interact with Gpi8p. Co-immunoprecipitation approaches were used to identify other interacting partners. With this approach the region of Gaa1p that interacts with Gpi8p was determined, and two other components of the transamidase complex (PIG-S, PIG-T) were identified. We also determined that the complex associates with tubulin and that this interaction is due to a short amino-terminal stretch of amino acids in Gaa1p. PHOSPHOLIPID FLIPPASES: Glycerophospholipids are synthesized on the cytoplasmic face of biogenic membranes and initially located in the cytoplasmic leaflet of the bilayer. The lipids must translocate across the bilayer to populate the exoplasmic leaflet for membrane assembly. Studies based on a detergent-solubilization-biochemical-reconstitution approach show that specific proteins in mammalian ER and bacterial cytoplasmic membranes catalyze flip-flop. We have extended these studies to develop assays using natural phospholipids (not phospholipid analogs as used previously), and to identify flippase-enriched protein fractions from detergent extracts of bacterial or endoplasmic reticulum. Further studies include analyses of Mycoplasma bovis, an organism with a limited repertoire of membrane proteins - we have shown that mycoplasma membranes have flippase activity and we fractionated detergent extracts of mycoplasma membranes to identify a membrane protein fraction that is enriched in flippase activity. STEROL TRANSPORT: The mechanisms involved in the transport of sterols from their site of synthesis to the plasma membrane are unknown. Using yeast as a model system, we are investigating these mechanisms. We have developed a cyclodextrin-based assay that allows us to measure ER to plasma membrane sterol transport in yeast, and have also laid the groundwork for transport studies by testing metabolic labeling procedures, chromatographic separations of sterols, and characterization of the metabolically radiolabeled sterol products. Our data indicate that the vesicle fusion protein sec18p and sec1p, and the vesicle coat protein sec12p, are not required for sterol delivery to the plasma membrane.

Impacts
We have studied a range of lipid structures from the point of view of their biosynthesis and intracellular transport. The results of our work have broad implications for understanding of cellular membrane lipid assembly.

Publications

Field, M.C., and Menon, A. K. (2001) Glycosylphosphatidylinositol membrane-anchored proteins (invited chapter). The Encyclopedia of Molecular Medicine (editor, T. E. Creighton; publisher, Wiley & Sons, Inc.), pp. 1497-1501.
Li, J., Rancour, D. M., Allende, M. L., Worth, C. A., Darling, D. S., Gilbert, J. B., Menon, A. K., and Young, W. W. (2001) The DXD motif is required for GM2 synthase activity but is not critical for nucleotide binding. Glycobiology 11, 217-229.
Vidugiriene, J., Vainauskas, S., Johnson, A. E., and Menon A. K. (2001) Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system. Eur. J. Biochem. 268, 2290-2300.
Baumann, N. A., and Menon, A. K. (2002) Lipid Modifications of Proteins. In "Biochemistry of Lipids, Lipoproteins and Membranes", Vance, D. E., and Vance, J., eds. (Elsevier, Amsterdam, The Netherlands), Chapter 2, in press.
Watkins, W. E. and Menon, A. K. (2002) Reconstitution of phospholipid flippase activity from E. coli inner membrane: a test of the protein translocon as a candidate flippase. Biol. Chem. in press.
Kubelt, J., Menon, A. K., Muller, P., and Herrmann, A. (2002) Transbilayer movement of flourescent phospholipid analogs in the cytoplasmic membrane of E. coli. Biochemistry, in press.
Menon, A. K. (2002) Lipid transport - an overview (editorial). Seminars in Cell and Developmental Biology 13, in press.

Progress 01/01/00 to 12/31/00

Outputs
We are interested in aspects of cellular membrane biogenesis concerned with lipid synthesis and intracellular transport. Our research is focussed on [1] assembly of glycosyl-phosphatidylinositols (GPIs) and GPI-anchored proteins, [2] phospholipid flip-flop in biogenic membranes, and [3] sterol transport from the ER to the plasma membrane. GPI ANCHORING OF PROTEINS AND INTRACELLULAR TRAFFICKING OF GPIs GPIs represent a family of glycolipids that exist in all eukaryotic cells and serve to anchor a variety of proteins to cell membranes. In collaboration with Art Johnson (Texas A& M University) we analyzed the GPI anchoring of a model protein using photocrosslinking techniques. These studies represented an initial exploration of the protein biochemical environment encountered by a pro-protein during its conversion to a GPI-anchored protein. They also yielded the first direct evidence that genetically identified components of the anchoring machinery are in close proximity to the pro-protein, and indicated the presence of additional protein subunits in the anchoring machinery. In a separate series of experiments to develop an assay for GPI-anchoring that could be used in an attenpt to purify the GPI anchoring enzyme, we discovered that one of the subunits (Gpi8p) of the anchoring enzyme is a soluble protein in trypanosomes. This has pointed us to the possibility of obtaining a crystal structure of this subunit and we are developing methods to obtain sufficient quantities of the protein for this purpose. PHOSPHOLIPID FLIPPASES Glycerophospholipids are synthesized on the cytoplasmic face of biogenic membranes and initially located in the cytoplasmic leaflet of the bilayer. The lipids must translocate across the bilayer to populate the exoplasmic leaflet for membrane assembly. We recently completed studies, based on a detergent-solubilization-biochemical-reconstitution approach, that showed that specific proteins in mammalian ER and bacterial cytoplasmic membranes catalyze flip-flop. We also attempted to identify flippase candidates in sequenced genomes. By applying a number of criteria we identified 4 genes in Escherichia coli that appeared to be plausible candidates. We are testing the products of these genes for flippase activity. STEROL TRANSPORT The mechanisms involved in the transport of sterols from their site of synthesis to the plasma membrane are unknown. We are interested in understanding these mechanisms using yeast as a model system. We have developed subcellular fractionation procedures that allow us to measure ER to plasma membrane sterol transport in yeast, and we have also laid the groundwork for transport studies by testing metabolic labeling procedures, chromatographic separations of sterols, and characterization of the metabolically radiolabeled sterol products. We have tested our experimental procedures in pulse-chase experiments and by analyzing the vesicular-transport dependent maturation of mannosylated inositol sphingolipids. Our data indicate that the `vesicle fusion protein' sec18p, and the vesicle coat protein sec12p, are not required for sterol delivery to the plasma membrane.

Impacts
We have studied a range of lipid structures from the point of view of their biosynthesis and intracellular transport. The results of our work have broad implications for understanding of cellular membrane lipid assembly.

Publications

Baumann, N.A., Vidugiriene, J., Machamer, C.A., and Menon, A.K. 2000. Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells. J. Biol. Chem. 275, 7378-7389.
Baumann, N.A., Menon, A.K., and Rancour, D.M. 2000. Functions of Glycosylphosphatidylinositols (invited chapter). In "Oligosaccharides in Chemistry and Biology: a Comprehensive Handbook", Ernst, B., Hart, G.W., and Sinay, P., eds. (Wiley/VCH, Weinheim, Germany), pp. 757-769.
McConville, M.J., and Menon, A.K. 2000. Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (invited review). Molecular Membrane Biology, 17, 1-16.
Menon, A.K., Watkins, W.E., and Hrafnsdottir, S. 2000. Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum membrane. Current Biology, 10, 241-252.
Hrafnsdottir, S., and Menon, A.K. 2000. Reconstitution and partial characterization of phospholipid flippase activity from detergent extracts of Bacillus subtilis cell membrane. J. Bacteriol., 182, 4198-4206.
Kostova, Z., Rancour, D.M., Menon, A.K., and Orlean, P. 2000. Photoaffinity labeling with P3-(4-azidoanilido)uridine 5-triphosphate identifies Gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyzes formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis. Biochem. J., 350, 815-822.
Sharma, D.K., Hilley, J., Bangs, J.D., Coombs, G.H., Mottram, J., and Menon, A.K. 2000. Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of soluble endoplasmic reticulum proteins. Biochem. J., 351, 717-722.

Progress 01/01/99 to 12/31/99

Outputs
We are interested in aspects of cellular membrane biogenesis concerned with lipid synthesis and intracellular transport. Our research is focussed on [1] assembly of glycosyl-phosphatidylinositols (GPIs) and GPI-anchored proteins, and intracellular transport of GPIs from the ER to the plasma membrane, [2] phospholipid flip-flop in biogenic membranes, and [3] sterol transport from the ER to the plasma membrane. GPI ANCHORING OF PROTEINS AND INTRACELLULAR TRAFFICKING OF GPIs: GPIs represent a family of complex glycolipids that exist in all eukaryotic cells and serve to anchor a variety of proteins to cell membranes. In collaboration with Art Johnson (Texas A&M University) we analyzed the membrane translocation and GPI anchoring of a model protein using photocrosslinking techniques. These studies yielded insight into the membrane disposition of the protein at various stages of its processing and will likely help to identify candidate ER resident proteins (including the GPI transamidase) involved in these processing events. In a parallel study, we developed a simple assay for the GPI anchoring reaction, and used the assay to prove explicitly that the anchoring event occurs via a transamidation mechanism. We recently completed an analysis of the subcellular distribution of free GPIs in mammalian cells. These analyses indicate that GPIs are transported from the ER to other cellular membranes, eventually concentrating in the exoplasmic leaflet of the plasma membrane. Work is currently in progress to understand the transport proces in more detail. PHOSPHOLIPID FLIPPASES: Glycerophospholipids are synthesized on the cytoplasmic face of biogenic membranes and initially located in the cytoplasmic leaflet of the bilayer. The lipids must translocate across the bilayer to populate the exoplasmic leaflet for membrane assembly. We recently completed studies, based on a detergent-solubilization-biochemical-reconstitution approach, that showed that specific proteins (flippases) in mammalian ER and bacterial cytoplasmic membranes are responsible for catalyzing flip-flop. We are in the process of developing a screen, using radiolabeled fluorescent phospholipid analogs as reporters, for the identification of bacterial mutants defective in flippase activity. STEROL TRANSPORT: The mechanisms involved in the transport of sterols from their site of synthesis to the plasma membrane are unknown. We are interested in understanding these mechanisms using yeast as a model system. To this end we have developed subcellular fractionation procedures that allow us to measure ER to plasma membrane sterol transport in yeast, and we have also laid the groundwork for transport studies by testing metabolic labeling procedures, chromatographic separations of sterols, and characterization of the metabolically radiolabeled sterol products. Preliminary data indicate that the `vesicle fusion protein' sec18p is not required for sterol delivery to the plasma membrane.

Impacts
We have studied a range of lipid structures from the point of view of their biosynthesis and intracellular transport. The results of our work have broad implications for understanding of cellular membrane lipid assembly.

Publications

Menon, A.K., and Butikofer, P. 1999. Biosynthesis of GPIs in mammalian cells. In "GPI-Anchored Membrane Proteins and Carbohydrates," Hoessli, D.C., and Ilangumaran, S., eds. (R.G. Landes Co., Austin, TX), pp. 15-28.
Puglielli, L., Mandon, E.C., Rancour, D.M., Menon, A.K., and Hirschberg. C.B. 1999. Identification and purification of the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter. J. Biol. Chem. 274:4474-4479.
Vidugiriene, J., Sharma, D.K., Smith, T.K., Baumann, N.A., and Menon, A.K. 1999. Segregation of GPI biosynthetic reactions in a subcompartment of the endoplasmic reticulum. J. Biol. Chem. 274:15203-15212.
Sharma, D.K., Vidugiriene, J., Bangs, J.D., and Menon, A.K. 1999. A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes: demonstration of a transamidation reaction mechanism. J. Biol. Chem. 274:16479-16486.

Progress 01/01/98 to 12/31/98

Outputs
Progress has been made on four fronts. (1) Transbilayer movement of phospholipids is required for membrane biogenesis. It has long been hypothesized that specific protein(s) - termed flippases - in the endoplasmic reticulum of eukaryotic cells and the cytoplasmic membrane of bacteria are able to facilitate this otherwise thermodynamically unfavorable transport process. There has been very little progress towards the identification of these flippases thus far. We now report the successful reconstitution of flippase-active proteoliposomes from detergent extracts of endoplasmic reticulum and bacterial membranes. The activity is reduced upon treatment of the protease treatment of the proteoliposomes. Furthermore, treatment of the detergent extract with sulfhydryl alkylating reagents prior to reconstitution reduces activity. These results point to protein involvement in the phospholipid translocation process and provide the methodology with which to isolate the protein. (2) We have synthesized photoaffinity reagents that mimic the substrates of enzymes and transporters required for protein and lipid glycosylation. These reagents have been used to identify a single polypeptide within the heterotetrameric complex involved in the first step of the biosynthesis of glycosylphosphatidylinositols, a ubiquitous family of eukaryotic glycolipids. The reagents have also assisted in the purification af a new sugar nucleotide transporter from rat liver Golgi membranes. (3) We have set up the methodology required for tha analysis of sterol trafficking in eukaryotes. Specifically we have characterized sterol metabolites that can be metabolically radiolabeled from radioactive methionine and radioactive acetate in the yeast Saccharomyces cerevisiae, and also characterized a subcellular fractionation procedure from which we obtain relatively pure yeast plasma membrane. With these procedures in hand, we are now in a position to determine the mechanisms by which sterols traffic from their site of synthesis, the endoplasmic reticulum, to the membrane where they are most concentrated in the cell, the plasma membrane. (4) In studies of the biosynthesis and trafficking of glycosylphosphatidylinositols (GPIs), we have determined that GPI biosynthesis is topographically heterogeneous in animal cells with post-initiation biosynthetic reactions being confined to a subcompartment of the endoplasmic reticulum. We have also determined that GPIs, after synthesis in the endoplasmic reticulum, can escape this membrane compartment and take one of two routes to the plasma membrane. Other GPIs are covalently attached to proteins bearing the appropopriate signal sequence and we have demonstrated that the reaction mechanism involved in this protein modification is a transamidation.

Impacts
We have studied a range of lipid structures from the point of view of their biosynthesis and intracellular transport. The results of our work have broad implications for understanding of cellular membrane lipid assembly.

Publications

Menon, A.K., and Butikofer, P. (1998) Biosynthesis if GPIs in mammalian cells. In "GPI-Anchored Biomolecules", Hoessli, D.C., and Ilangumaran, S., eds. (R.G. Landes Co., Austin, TX).
Menon, A.K. (1998) Lipids: more than just membrane fabric. Trends Cell Biol. 8, 374-376.
Puglielli, L., Mandon, E.C., Rancour, D.M., Menon, A.K., and Hirschberg, C.B. (1999) Identification and purification of the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter. J. Biol. Chem. 274, 4474-4479.
Rancour, D.M., and Menon, A.K. (1998) Identification of endoplasmic reticulum proteins involved in glycan assembly: Synthesis and characterization of 4-azidoanilido-UTP, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins. Biochemical J. 333, 661-669.
Sharma, D.K., Vidugiriene, J., Bangs, J.D., and Menon, (1999) A.K. A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes: demonstration of a transamidation reaction mechanism. J. Biol. Chem. submitted.
Vidugiriene, J., Sharma, D.K., Smith, T.K., Baumann, N.A., and Menon, A.K. (1999) Segregation of GPI biosynthetic reactions in a subcompartment of the endoplasmic reticulum. J. Biol. Chem. in press.
Rancour, D.M., Baumann, N.A., and Menon, A.K. (1999) Functions of Glycosylphosphatidylinositols (invited chapter). In "Saccharides in Chemistry and Biology: a Comprehensive Handbook", Ernst, B., Hart, G.W., and Sinay, P., eds. (Wiley/VCH, Weinheim, Germany). submitted.

Progress 01/01/97 to 12/31/97

Outputs
This project is concerned with the biosynthesis and intracellular transport of simple and complex lipids. We have shown that glycosylphosphatidylinositols (GPIs), an ubiquitous class of eukaryotic glycolipids, are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). GPIs can then be flipped across the ER membrane to be covalently linked to freshly translocated proteins bearing an appropriate carboxyl-terminal signal sequence. The bulk of the GPI molecules are distributed amongst other cellular membranes, including the plasma membrane. The redistribution most likely occurs by non-vesicular means possibly via lipid transfer proteins or at points of membrane contact. It is likely that GPIs, unlike other glycolipids, are located in the cytoplasmic leaflet of the plasma membrane. In separate experiments we have begun to develop methodology to identify glycosyltransferase of the ER membrane that are involved in the synthesis of GPIs, dolichol-linked saccharides, and other glycoconjugates. We have synthesized a credible photoaffinity reagent that can be used to tag these glycosyltransferases and so aid in their purification. We are also working to identify the machinery involved in transbilayer movement of phospholipids in the endoplasmic reticulum and in the cytoplasmic membrane of bacteria. These movement or flip-flop is essential for membrane biogenesis as it permits the assembly of the membrane bilayer. Flip-flop is presumed to be mediated by proteins called flippases, but no biogenic membrane flippase has been identified. We have developed a highly time-resolved assay to measure flippase activity and we are in the process of attempting to reconstitute the activity from detergent-solubilized membranes.

Impacts
(N/A)

Publications

Butikofer, P., Boschung, M., and Menon, A.K. 1995. Production of a nested set of glycosylphosphatidylinositol structures from a glycosylphosphatidylinositol-anchored protein. Anal. Biochem. 229, 125-132.
Menon, A.K. 1995. Flippases. Trends Cell Biol. 5, 355-360.
publications 1994-present
Menon, A.K. 1994. Characterization and structural analysis of GPI anchors. In Guide to Techniques in Glycobiology (Methods in Enzymology, Vol. 230), Lennarz, W.J. and Hart, G.W., eds. (Academic Press, New York, NY), pp. 418-442.
Vidugiriene, J., and Menon, A.K. 1994. Topology of GPI biosynthesis in the endoplasmic reticulum. Brazilian J. Med. Biol. Res. 27, 167-175.
Vidugiriene, J., and Menon, A.K. 1994. The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum. J. Cell Biol., 127, 333-341.
Vidugiriene, J., and Menon, A.K. 1995. Biosynthesis of glycosylphosphatidylinositol anchors. In Lipid Modifications of Proteins (Methods in Enzymology, Vol. 250), Casey, P.J., and Buss, J.E., eds. (Academic Press, New York, NY), pp. 513-535.
van't Hof, W., Rodriguez-Boulan, E., and Menon, A.K. 1995. Non-polarized distribution of glycosylphosphatidylinositols in the plasma membrane of Madin Darby canine kidney cells. J. Biol. Chem. 270, 24150-24155.
Vidugiriene, J., and Menon, A.K. 1995. Soluble constituents of the ER lumen are required for GPI anchoring of a model protein. EMBO J. 14, 4686-4694.
Butikofer, P., Boschung, M., Brodbeck, U., and Menon, A.K. 1996. Phosphatidylinositol hydrolysis by Trypanosoma brucei GPI phospholipase C. J. Biol. Chem., 271, 15533-15541.
Hrafnsdottir, S., Nichols, J.W., and Menon, A.K. 1997. Transbilayer movement of fluorescent phospholipids in Bacillus megaterium membrane vesicles. Biochemistry, 36, 4969-4978.
Menon, A.K., Baumann, N.A., van't Hof, W., and Vidugiriene, J. 1997. Glycosylphosphatidylinositols: biosynthesis and intracellular transport. Biochem. Soc. Trans. 25, 861-865.
Rancour, D.M., and Menon, A.K. 1998. Identification of endoplasmic reticulum proteins involved in glycan assembly: Synthesis and characterization of 4-azidoanilido-UTP, a membrane-topological photoaffinity probe for uridine diphosphate-sugar binding proteins. Biochemical Journal, in press

Progress 01/01/95 to 12/30/95

Outputs
We are studying the general problem of membrane biogenesis in eukaryotes and prokaryotes. Our research efforts are focussed on (i) the biosynthetic routes involved in the construction of complex membrane lipids, particularly the glycosylated phosphatidylinositols found in all eukaryotes and (ii) the mechanisms that are responsible for transporting simple and complex lipids and sterols across membranes and between sub-cellular organelles. The long-term aim is to describe these pathways in molecular detail, and to isolate the carriers and enzymes involved. Experimental work will involve the use of protozoa, bacteria and cultured animal cells, as well as tissues such as liver. We do not plan to experiment directly with animals except to obtain biological material. A range of techniques such as biosynthetic and cell-free radiolabeling, sub-cellular fractionation, membrane solubilization and reconstitution, protein purification and organic synthesis will be used.

Impacts
(N/A)

Publications

Vidugiriene, J. and Menon, A. K. (1995) Biosynthesis of glycosylphosphatidyl-inositol anchors. In: Lipid Modifications of Proteins, Methods in Enzymology, Vol. 250, pp. 513-535, edited by P. J. Casey and J. E. Buss (Academic Press, New York
Btikofer, P., Boschung, M. and Menon, A. K. (1995) Production of a nested set of glycosylphosphatidylinositol structures from a glycosylphosphatidylinositol-anchored protein. Anal. Biochem. 229, 125-132.
Menon, A. K. (1995) Flippases. Trends in Cell Biol. 5, 355-360. van't
Hof, W., Rodriguez-Boulan, E. and Menon, A. K. (1995) Nonpolarized distribution of glycosylphosphatidylinositols in the plasma membrane of polarized Madin-Darby canine kidney cells. J. Biol. Chem. 270, 24150-24155.
Vidugiriene, J. and Menon, A. K. (1995) Soluble constituents of the ER lumen are required for GPI anchoring of a model protein. EMBO J. 14, 4686-4694.