Cornell University Weill Medical College""The Menon Lab
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Current Research Program

We are interested in mechanisms of intracellular lipid transport. While the transport of proteins across and between membranes has been intensively and successfully studied since the 1970s, little is known about how lipids are trafficked within the cell. Two examples serve to highlight major gaps in our understanding of this aspect of cell physiology. N-glycosylation of proteins, an essential post-translational modification in eukaryotes, requires the transbilayer flip-flop of three different glycolipids across the endoplasmic reticulum (ER) membrane. The mechanism by which lipids are flipped across the ER membrane is unknown except that it is ATP-independent and requires specific (as yet unidentified) proteins. Sterols are synthesized in the ER and transported to the plasma membrane (PM) where they constitute almost half the membrane lipids. Transport occurs by an ATP-dependent, non-vesicular mechanism about which nothing is known. We are interested in both these problems – details are provided below. Other projects in the lab continue our long-standing interest in the biosynthesis of glycosylphosphatidylinositol (GPI) anchored proteins.  

ER lipid flip-flop: searching for flippases

Lipids diffuse rapidly in the plane of the membrane, but their transverse diffusion - movement from one side of the bilayer to the other, or flip-flop - is intrinsically very slow and does not occur on a physiologically relevant time scale. This is because the hydrophobic interior of the membrane presents a considerable energy barrier to the transit of the polar lipid headgroup.  For common phospholipids such as phosphatidylcholine (PC), this barrier is estimated at ~20-50 kcal/mol, roughly equivalent to the energy derived from hydrolysis of 3-7 ATP molecules. For Man5GlcNAc2-PP-dolichol (M5-DLO), a glycolipid intermediate in the pathway of protein N-glycosylation, the barrier to spontaneous flip-flop is much higher (>130 kcal/mol). Lipid flip-flop is essential for numerous physiological processes and occurs rapidly in many different biomembranes as a result of the action of transport proteins. We are interested in ATP-independent lipid transporters (flippases) in the endoplasmic reticulum (ER) that play an essential role in the biosynthesis of glycolipid precursors of essential cell surface glycoconjugates such as N-glycosylated proteins, GPI-anchored proteins and glycosphingolipids. In each case, the glycolipid precursor is assembled via a multi-step pathway that starts in the cytoplasmic leaflet of the ER and continues in the lumenal leaflet, necessitating flipping of lipid intermediates such as M5-DLO, mannose-phosphate-dolichol, glucose-phosphate-dolichol, glucosylceramide and glycosylphosphatidylinositols (GPIs)(see figure 1). In addition to possessing glycolipid transporters, the ER also has a glycerophospholipid flippase activity; this is required to balance the phospholipid content of the two leaflets of the bilayer since phospholipid biosynthesis occurs on the cytoplasmic face of the ER.
None of the ER flippases have been identified at the molecular level. We have developed methods to assay flip-flop of glycerophospholipids as well as isoprenoid-based glycolipids in biochemically reconstituted systems (see Frank et al. (2008) Nature 454:E3; Sanyal et al. (2008) Biochemistry 47:7937; Sanyal and Menon (2009) PNAS 106:767). We are using these methods to identify ER flippases by protein purification approaches as well as by screening of systematic collections. Our aim is to understand how they work. We collaborate with Prof. Brian Chait of the Rockefeller University on aspects of this project.

How are sterols transported between the ER and plasma membrane?

We are interested in learning how newly synthesized sterols are transported from the ER where they are synthesized, to the plasma membrane (PM). Sterols are unevenly distributed in the endomembrane system of eukaryotic cells. They are 10-fold more highly concentrated in the PM, where they restrict the flexibility of lipid acyl chains, thicken the PM and make it less permeable to small molecules.  Sterol concentration in the ER is low, consistent with the need for a more fluid membrane to accommodate processes such as membrane protein translocation. 
Sterol transport between the ER and PM occurs primarily by a non-vesicular mechanism that equilibrates the sterol content of the two compartments. This is possible despite the ~10-fold sterol concentration difference between the ER and PM since specific features of the PM lower the chemical activity of sterol to a point where it is equal to that of the ER: the average chemical activity coefficient of the PM sterol pool is likely to be ~10-fold lower than that of the ER sterol pool (see figure 2).
Our current efforts (all using Bakers’ yeast, Saccharomyces cerevisiae) are directed toward [i] analyses of the role, in sterol equilibration, of the sterol-binding Osh (homologs of mammalian oxysterol binding proteins) proteins, some of which appear to be capable of bridging the ER and PM (in collaboration with Chris Beh, Simon Fraser University, British Columbia, Canada ( [ii] using fluorescence methods to analyze the transbilayer distribution of sterol at the PM, and [iii] using a genome-wide approach to identify yeast mutants with defects in sterol transport. This latter approach is expected to reveal the molecular identity of components of the protein machinery involved in sterol transport. See Sullivan et al. (2009) Eukaryot. Cell 8:161 for a description of a genetic selection to identify sterol transport mutants. See also Maxfield and Menon (2006) Curr. Opin. Cell Biol. 18:379.

GPI anchoring of secretory proteins

Glycosylphosphatidylinositols (GPIs) are a family of complex glycolipids that exist in all eukaryotic cells and serve to anchor a variety of proteins to cell membranes; roughly 1% of all proteins encoded by eukaryotic genomes are post-translationally modified at their C-terminus by GPI. Examples include acetylcholinesterase, folate receptor and the prion protein. GPI biosynthesis is critical for normal cell growth and perturbed in human cancers and a number of genetic diseases such as paroxysmal nocturnal hemoglobinuria, an acquired hemolytic disease. Genetic abrogation of GPI biosynthesis results in embryonic lethality in mammals. GPI-proteins are critical for the viability of a parasitic protozoa and fungi and the GPI assembly pathway has been validated as a therapeutic target for protozoal and fungal diseases.
GPIs are synthesized in the ER and subsequently attached to proteins through a transamidation reaction in which a complete GPI structure is covalently linked to an ER-translocated, membrane-integrated protein containing a carboxyl-terminal GPI-directing signal sequence (see figure 3). Our laboratory has been historically associated with delineating steps of the ER-localized GPI biosynthetic pathway and describing the topological complexity of the assembly sequence.
We are interested in structure-function analyses of GPI transamidase, the multi-subunit enzyme complex required to attach a GPI anchor to proteins. GPI transamidase consists of five membrane proteins, only one of which (Gpi8) has sequence homology to other known proteins.  The other subunits are required for enzyme function in vivo, but sequence gazing offers no insight into the possible role of these proteins. We are interested in knowing the spatial arrangements of subunits within each complex, identifying subunits responsible for binding substrates, and determining functionally relevant domains within each of the subunits. Our studies are done with mammalian cells as well as with the protozoan parasite Trypanosoma brucei whose GPIT has a somewhat different subunit composition compared with the mammalian enzyme. See Vainauskas and Menon (2006) J. Biol. Chem. 281:38358 and Orlean and Menon (2007) J. Lipid Res. 48: 993 for more information.

The mechanism of action of a flippase may be likened to that of swiping a card through a card readerFIGURE 1 Lipid flip-flop across the ER during assembly of the oligosaccharide-PP-dolichol precursor of protein N-glycans. Glc3Man9GlcNAc2-PP-dolichol, the glycan donor in protein N-glycosylation, is synthesized in a multi-step pathway in the ER. The first 7 steps, leading to the synthesis of M5-DLO from dolichol-P occur on the cytoplasmic face of the ER. GDP-mannose and UDP-GlcNAc directly contribute the sugar moieties for these reactions. M5-DLO is then flipped into the ER lumen by M5-DLO flippase, and extended in a further 7 reactions to yield Glc3Man9GlcNAc2-PP-dolichol. The sugar donors for these reactions are (mannose-phosphate dolichol) MPD and glucose-phosphate dolichol (GPD). MPD is synthesized from dolichol-P and GDP-mannose on the cytoplasmic face of the ER, then flipped to the ER lumen by MPD flippase. In the lumen it is the mannosyl donor for the 4 reactions that convert M5-DLO to Man9GlcNAc2-PP-dolichol. GPD is synthesized from dolichol-P and UDP-glucose on the cytoplasmic face of the ER, then flipped to the ER lumen by GPD flippase.

The mechanism of action of a flippase may be likened to that of swiping a card through a card readerFIGURE 2 Model for the maintenance of sterol distribution between the ER and the PM. Sterols are trafficked between the ER and PM by an equilibrative, non-vesicular mechanism that nevertheless preserves the high concentration of sterol in the PM. This can be explained by considering the chemical potential (m) or chemical activity (a) of sterol in the membrane, rather than its concentration. At equilibrium, the chemical activity of sterol in the ER (aER) and PM (aPM) would be the same, even though the sterol concentration in the two membranes is different (cER << cPM). This can be achieved by lowering the chemical activity coefficient (g; g = a/c) of a large proportion of the PM’s sterol content through association with sphingolipids or other phospholipids with saturated acyl chains. This would create two pools of sterol (pool 1, ‘high g’; pool 2, ‘low g’) in the PM as shown in the model figure. The two pools would be at equilibrium and thus have the same activity (a1 = a2 = aPM) but different chemical activity coefficients (g1 > g2) and concentrations (c1 < c2). In this fashion, the PM can sequester much more sterol than the ER.

The mechanism of action of a flippase may be likened to that of swiping a card through a card readerFIGURE 3 GPI anchoring of proteins. ER-translocated proteins with a C-terminal GPI signal sequence are recognized by GPIT on the lumenal face of the ER. The C-terminal signal sequence is cleaved between residues w & w+1 and replaced with GPI. GPI is attached to the w residue by an amide bond between the a-COOH of w and the NH2 of the capping ethanolamine-phosphate (EtN-P) residue in GPI.

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