The cell fractionation of rat intestinal cells has revealed differences between the lipid content of their Ap and Bl surfaces (Hauser 1 9 8 0 ). A 2 -4 fold increase of glycosphingolipids was evident in their Ap domain, with a concomitant equivalent increase in phosphatidylcholine in their Bl domain. Non-polarised lipids such as phosphatidylethanolamine and cholesterol are found evenly distributed between the two domains. Membrane sampling of the Ap (by Influenza budding) and Bl (VSV budding) surfaces of MDCK cells reveals similar lipid polarity to rat intestinal cells (McClosky 1 9 8 4 ) and CaCo2 cells (van't Hof 1 9 9 0 ). These observations predict a conserved mechanism for epithelial membrane lipid sorting.
The glycosphingolipids in the exoplasmic leaflet completely cover the Ap domain and face the lumenal environment. Sphingolipids are particularly resistant to bile salt detergents and the lipid hydrolase, phospholipase A2, which is especially abundant in the intestinal lumen. Therefore, a potential protective role against the lumen is offered by sphingolipids.
I . 3 . C . Lipid transport
The fluorescent analogues; C6-NBD-glucosylceramide and C6- NBD sphingomyelin, have been used to study lipid delivery to the PM (under conditions that prevent endocytosis of the markers from the PM, by back-exchange using either BSA [van Meer 19 86 ] or liposomes [van Meer 1 9 8 7 ] to remove the lipids from the PM). Sorting appears to occur at the TGN, with polarised transport to the PM in 1 hour. Trafficking is most likely to be mediated by a tubulo-vesicular mechanism, as suggested by the lumenal location of sphingomyelin. More direct proof (va n 't Hof 1 9 9 0 ) has been offered by the observation th a t during mitosis, when vesicular transport is interrupted, so too is the transport of sphingolipids. However, the transport of phosphatidylethanolamine (which can be transported via cytoplasmic exchange by phospholipid transfer proteins) continues.
1.3.d. A role for lipids in protein sorting
The observed lipid polarity in epithelial cells could be generated by physical separation of components in the TGN. It is known that sphingolipids, especially glycosphingolipids, have a tendency to associate by hydrogen bonding and it has been proposed (Simons 1 9 8 8 ) that they cluster into microdomains in the TGN, facilitated by local conditions (e.g. low pH).
Glycoprotein and glycolipid sorting exhibit similar transport kinetics (Wattenberg 1 9 9 0 ) and both can be blocked at 20^C. It is therefore possible th at they are transported together. Protein microdomains have been observed in the TGN (Geuze 1 9 8 7 ) and it is therefore possible th at proteins and lipids may interact in this compartment and subsequently be co-sorted (Simons 1985, 1987, 19 88 ). Such lipid and protein association has been illustrated by the presence of cholesterol microdomains (caveolae) into which GPI- anchored proteins cluster at the PM (Rothberg 1 9 9 0 ). A similar interaction at the TGN may be involved in the Ap sorting mechanism (See 'GPI-anchored proteins').
1.3 .e . Maintenance of lipid polarity
Continual membrane tra ffic jeopardises the integrity of cell polarity. The potential losses and gains of both proteins and lipids resulting from membrane tra ffic must be counterbalanced. Redistribution of proteins and lipids may be attained by vesicular transport, however, redistribution of membrane (misplaced lipids) may be a more urgent requirement. Lipid redistribution may be achieved in four ways: Firstly, lipids may recycle in vesicles low in protein content. Secondly, misplaced lipids may be degraded. Thirdly, delivery of vesicles occurs via intermediates, such that the vesicle matures gradually into the membrane of its target. Lastly, the lipids may retain their membrane composition by cytosolic lipid transfer.
Mitochondria and peroxisomes obtain their proteins and lipids from mechanisms that are independent of the secretory pathway. Their lipids are obtained by cytosolic phospholipid exchange proteins, extracting phospholipids from the 'lipid-rich' ER to the
lipid-poor mitochondria and peroxisomes, whilst proteins are imported post-translationally from the cytoplasm.
In the secretory pathway, forward transport of newly synthesised proteins and lipids appear to be conducted by vectorial movement (van't Hof 1 9 9 0 ). However it is possible that lipid recycling might occur primarily by cytosolic exchange (Bankaitis 1 9 9 0 ), with protein recycling occurring vectorially.
Obviously cytosolic exchange can only occur from the cytosolic leaflet, disproportionate loss of membrane may be controlled by the 'flipping' of some lipids from the exoplasmic to the cytoplasmic leaflet. Some ATP-dependent cell surface 'flippases' do exist that translocate phosphatidylethanolamine and phosphatidylserine (but not phosphatidylcholine and sphingomyelin) into the cytoplasmic leaflet (Bankaitis 1 9 9 0 ). Similar specific 'flippases' could act along the secretory pathway, thus maintaining the bilayer and providing certain lipids with the opportunity to recycle to 'lipid-poor' membranes.
The observed concentration gradient of lipids from the ER to the Golgi (Table 3) (Simons 1 9 88 ) is consistent with the hypothesis that some lipids preferentially recycle, whilst others cannot. Lipids that are concentrated towards the ER presumably recycle via cytosolic transfer, whilst those that cannot become concentrated towards the PMs. In fact seel 4p, a phospholipid transfer protein located within the yeast Golgi has been shown to be essential for Golgi to PM transport (Cleves 1 9 9 1 ), which may indicate a specific requirement for a high ratio of phosphatidylinositol/phosphatidylcholine. Phosphatidylethanolamine is present in both leaflets, is capable of flipping y e t is evenly distributed throughout the cell, thus some specificity of cytosolic transfer must exist.
Endoplasmic reticulum to Plasma membrane
Increasing [LIpId] Decreasing [Lipid]
glycosphingolipids (E)
sphingomyelin (E) phosphatidylcholine (E+C)
phosphatidylserine (E) phosphatidylinositol (C)
cholesterol (E+C)
Table 3. Lipid concentration gradient observed between the ER and piasma membrane. Upids can be found in either the exoplasmic leaflet (E) or the cytoplasmic leaflet (C), or both (E+C). Lipids found concentrated towards the PM (left-hand column) may be unable to undergo cytosolic transfer due to reduced residence in the cytoplasmic leaflet.
1.4.
Protein targetting in epithelial cells
Although it is not known how lipids achieve their Ap versus Bl distribution in polarised epithelial cells, extensive analysis of protein ta rg e ttin g by the molecular biological approach of mutagenesis and fusion protein production, has begun to identify signals required for polarised targetting.
1 . 4 . a. T argetting signals in HA and VSV G proteins
As previously discussed, influenza virus HA and VSV G are transported directly to the Ap and Bl membrane domains of epithelial ceils respectively (Misek 19 84 , Pfeiffer 1 9 8 5 ). Both HA and VSV G proteins are co-localised in the TGN (Fuller 1 9 8 5 ), after which sorting to their respective domains probably occurs via distinct transport vesicles (Wandinger-Ness 1 9 9 0 ). The polarised delivery of these glycoproteins does not require glycosylation (Roth 1 9 7 9 , Green 1 9 8 1 ). Both these proteins need to noncovalently associate into trimers before transport to the Golgi can be achieved. This q u a rte rn a ry s tru c tu re re q u ire m e n t has com plicated mutagenesis experiments.
The production of HA and VSV G protein chimaeras revealed that the ectodomain of HA (Roth 1987, Puddington 1987, McQueen 1986) or VSV G protein (McQueen 1 9 8 7 ) was required for Ap or Bl sorting respectively. Contradictory results have been obtained by the production of chimaeras with Friend murine leukaemia virus which suggest that the Ap signal in HA resides in the cytoplasmic domain. The contradictory and confusing results obtained could be due to a number of factors; such as the complications of trimérisation and the fact th a t analysis was carried out at steady-state by non- quantitative immunofluoresence on poorly polarised cells.
Recently, several normally Bl proteins have been converted to apically directed proteins by the mutation or deletion of their cytoplasmic domains. There appears to be a correlation between rapid endocytosis via clathrin-coated pits and Bl targetting, suggesting th at the signals could be the same or very closely related.
1.4 .b . Correlation between endocytosis and Bl targetting
A particularly revealing correlation between the endocytic and Bl signals was observed for Influenza HA, which is normally Ap and not endocytosed (Roth 19 86 ). After conversion of a single residue in its cytoplasmic domain (Cys 543 to a tyr 5 4 3), the intact trimerised
protein changes from an Ap non-endocytosing to a Bl endocytosing form (Brewer 1 9 9 1 ). Endocytosis of the mutant protein occurred efficiently via clathrin-coated pits in CVI (Lazarovits 1 9 8 8 ) and MDCK cells (Brewer 1 9 9 1 ). The tyr 5 4 3 mutant did not transiently appear at the Ap surface in MDCK cells, as determined by trypsin cleavage and antibody capture assays.
Another example is shown by two related Fc receptor isoforms, which differ in their ability to localise to clathrin-coated pits and have been shown to exhibit distinct polarities when transfected into MDCK cells (Hunziker 1 9 8 9 ), with coated pit and Bl locations correlating. The non-polarised isoform differs from the Bl form by a cytoplasmic 47 amino acid insert. Analysis of deletions of the cytoplasmic tail of the Bl receptor (Hunziker 1 9 8 9 ) revealed that endocytosis and Bl localisation were always coupled and the signal
responsible for these phenotypes resided within a 13 amino acid stretch, 18 amino acids from, the membrane. Interestingly, in contrast to the non-polarised isoform, these mutations exhibited 80-90% Ap localisation. It is possible that the displacement of the putative signal from the membrane has resulted in weakening of the signals functionality.
The human nerve growth factor receptor when transfected into MDCK cells, shows greater than 80% Ap localisation, as does a cytoplasmic tailless mutant (Le Bivic 1 9 9 1 ). However, the internal deletion of 57 amino acids from the cytoplasmic tail results in a tyrosine being positioned in an appropriate environm ent for endocytosis (Ktistakis 1 9 9 0 ) proximal to the membrane. This construct exhibited greater than 95% Bl localisation, suggesting th at new signals may be formed when placed in the appropriate context.
Igpl 2 0 is a major lysosomal membrane protein containing a short