The Superlattice Model of Membrane Lateral Organization Implications on Lipid Compositional Regulation and Maintenance of Organelle Boundaries

INTRODUCTION It is unknown how cells regulate the lipid composition of their membranes. Overexpression of the rate limiting biosynthetic enzymes does not increase significantly the concentration of the corresponding phospholipids. This is because (unknown) phospholipases are activated and rapidly degrade the phospholipid in excess, but it is not known what triggers/inhibits the activity of those phospholipases. The activities of both the synthetic enzymes and phospholipases must be closely coordinated to avoid futile competition Such coordination might be achieved via the action on some proteins that sense the concentration of the individual lipids, but such proteins have not been described so far We and others have proposed that the different lipid species in multicomponent membranes tend adopt regular (even) lateral distributions (Somerharju et al., 1999; Chong & Sugar, 2001). This putative phenomenon, referred to as lipid superlattice (SL) formation, predicts that there are several “critical”, energetically favorable compositions Such critical composition could play a key role in the regulation of the lipid composition of cellular membranes by inhibiting/activating synthetic enzymes and phospholipases in a coordinated manner The superlattice-based regulation mechanism is supported by that the phospholipid composition of the erythrocyte membrane falls were close to a critical composition predicted by the SL- model (Virtanen et al.1998). Intriguingly, the SL-model could also help to explain how cells maintain the integrity or their organelles. The model namely predicts that when a critical concentration of a phospholipid (or cholesterol) is exceeded (e.g. due to ongoing synthesis), segregation of domains with different SL-compositions takes place. Such segregation can drive membrane budding (Julicher & Lipowsky, 1993) and could thus be the basis of organelle integrity. Perhaps even the existence of separate Golgi compartments could be based on this principle?

Phospholipid composition of mammalian membranes are influenced by: A. Activity of rate-limiting synthetic enzymes Cytidyltransferase (influenced by e.g. PC content and membrane charge) PS-synthase (influenced by e.g. PS content or membrane charge) B. Activity of phospholipases Overexpression of Cytidyltransferase => PC does not increase (Walkey et al. 1994) Overexpression of Ethanolamine kinase => PE does not increase (Lydikis et al. 2001) Overexpression of PS-Synthase => PS does not increase (Stone et al. 1998) Increased amounts of degradation products in medium!

Synthesis ↑ PC ↓ degradation Synthesis ↑ PE ↓ Degradation PC-Regulator PE- Regulator ?? PS-Regulator PI- Regulator Synthesis ↑ PS ↓ Degradation Synthesis ↑ PI↓ Degradation Synthesis ↑ PC ↓ Degradation PC-Regulator What is the primary regulatory factor? A. Multiple regulatory proteins? ?? = How can all these enzymes be coordinated to avoid futile competition between synthetic and degradative machineries?

 Only a limited number of allowed compositions! Alternative model: Tendency of lipids to abopt regular distributions (i.e. superlattice formation) coordinates synthesis and degradation 15.4 % 25 % 50%

Superlattices are minimum energy structures (i.e. not permanent or rigid) because they..

1. Allow tightest packing of the lipids (Enthalpic effect) => 2. Avoid proximity of charged lipids (Coulombic effect) => 3. Increase rotational freedom of headgroups (Entropic effect) =>

Superlattice compositions are local energy minima and thus inherently more stable than other compositions Order/disorder transitions could activate/inhibit synthetic enzymes =>..and phospholipases as well => How could Superlattice formation control lipid compositions? Dissolved enzyme = active Aggregated enzyme = inactive

Supporting evidence: Phospholipid composition of erythrocyte membrane is compatible with the SL-model In 3-component superlattices the crical concentrations are Multiples of 11.1 mol% ! Assumption: Phospholipids can be divided into 3 classes: 1) Choline phospholipids (CP) = SM + PC Large headgroup 2) Ethanolamine phospholipids = PE Small headgroup 3) Acidic phospholipids (AP) = PS ( PI, PA) Negatively charged headgroup

Black = Experimental (Dogde & Phillips, J. Lipid Res. 8,667, 1967) Red = Predicted by SL-Model (Virtanen et. Al. PNAS 95, 4964, 1998) Outer Inner Cholesterol supplements PE as a headgroup spacer in OL? Superlattices also help to maintain transversal membrane asymmetry? Phospholipid composition of the erythrocyte membrane

SL-model and maintenance of organelle boundaries Domain segregation is known to drive budding and fission of vesicles in model systems (e.g., Julicher and Lipowsky 1993) SL-model predicts that domains with different SL-compositions do not mix Ongoing lipid synthesis in ER could result partial conversion of the ER-SL to another one resulting in segregation of domains and budding of those domains as vesicles forming the Golgi membranes The existence of several Golgi compartments could be based on subsequential segregation of different SL-domains. The stacks do not fuse because the different SLs do not like to mix The lipid composition of raft domains in the PM of nucleated cells could correspond to that of the erythrocyte membrane

Conclusions Experiments with model membranes and theoretical arguments suggest that lipids in natural membranes have a significant tendency to adopt regular distributions Such a tendency could play a key role in synchronizing lipid synthesis and degradation thus maintaining membrane lipid compositions Different organelle membranes could have different superlattices, which could help to maintain the integrity of the organelles, particularly those connected to each other via constant vesicle flow Rafts are proposed to have a superlattice organization which is similar to that suggested previously for the erythrocyte membrane REFERENCES Chong PL, Sugar IP. Fluorescence studies of lipid regular distribution in membranes. Chem Phys Lipids Jun;116(1-2): Julicher F, Lipowsky R. (1993) Domain-induced budding of vesicles. Phys Rev Lett. 70: Lykidis A, Wang J, Karim MA, Jackowski S (2001) Overexpression of a mammalian ethanolamine-specific kinase accelerates the CDP-ethanolamine pathway. J Biol Chem. 276: Somerharju, P., Virtanen, J.A. and Cheng, K.H. (1999) Lateral organisation of membrane lipids. The superlattice view. Biochim. Biophys. Acta 1440,32-48 Stone SJ, Cui Z, Vance JE. (1998) Cloning and expression of mouse liver phosphatidylserine synthase-1 cDNA. Overexpression in rat hepatoma cells inhibits the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis. J Biol Chem. 273: Virtanen, J. A., Cheng, K. H. and Somerharju, P. (1998) Phospholipid composition of the erythrocyte membrane can be rationalized by a lipid head group superlattice-model. PNAS 95, Walkey CJ, Kalmar GB, Cornell RB. (1994) Overexpression of rat liver CTP:phosphocholine cytidylyltransferase accelerates phosphatidylcholine synthesis and degradation. J Biol Chem. 269: