Membrane traffic mediates the exchange of components between the different cellular organelles. Membrane proteins and lipids are synthesized in the endoplasmic reticulum and from there are transported to their subcellular sites of action [109, 110]. While peripheral membrane proteins as well as single lipids bound to lipid transfer proteins can shuttle between different membranes via the cytoplasm or through contacts between membranes , most membrane turnover is mediated by vesicular traffic. Directed vesicular transport involves several regulated steps:
• lateral sorting of membrane components according to their destination (i.e., the concentration of cargo following the same pathway and its segregation from cargo following different pathways);
• stabilization of a membrane domain destined for trafficking;
• bending of the membrane domain into the shape of a vesicle or tubule;
• pinching off from the donor compartment;
• traffic through the cytoplasm by passive diffusion or motor-protein-mediated transport along microtubules or actin filaments;
• fusion with the acceptor compartment; and
The best-understood sorting mechanism for transmembrane proteins employs recyclable protein coats, such as clathrin-, COPI- or COPII-coats [111, 112] (Fig. 1.1, left panel). In this case the cargo proteins contain specific sorting signals in their cytoplasmic domains, which are bound by adaptor molecules, to which the coat proteins are recruited. Oligomerization of the coats leads to bending of the membrane domain into a vesicle, which is pinched off by the action of the GTPase dynamin and released into the cytosol. Here the coat disassembles, enabling the vesicle to fuse with its target membrane. This protein-driven mechanism operates by active inclusion of certain components and is not very efficient at excluding.
For other sorting events in membrane traffic, the lipid bilayer itself has been proposed to play the decisive role, and proteins only regulate what lipids can do on their own . From theoretical considerations and model membrane studies it is known that if phases with different properties coexist in the same membrane, then the mismatch of interactions at the phase boundary leads to the so-called "line tension" - the two-dimensional equivalent of surface tension. Multiplied with the length of the phase boundary it gives rise to the "line energy". One way to minimize line energy is therefore to minimize the contact between phases. In the case of domains in cell membranes, this can be achieved by fusion of many small domains into one large domain, and bending the domain out of the surrounding bulk membrane . The bending energy needed to curve the membrane as the domain buds out counteracts the line energy. As the bending energy increases and the line energy decreases, the domain reaches a stable curvature when the sum of the two energies is minimal. For small domains this can be when the domain is still connected to the bulk membrane, but above a critical domain size budding becomes energetically favorable. This mechanism is termed "domain-induced budding" (Fig. 1.1, right panel) and is initially achieved purely by lipid-driven phase separation . However, in order to attain directionality in the budding process (i. e., budding towards the cytoplasm in most cases in cells) and also kinetics that are compatible with the cell's needs, proteins will have to control this process.
The fact that lipids are unevenly distributed between the two surfaces, the apical and the basolateral membrane domains, of epithelial cells  together with the finding that newly synthesized glucosyl-ceramide upon leaving the Golgi complex becomes two- to three-fold enriched in the apical versus the basolateral plasma membrane , has led to the proposal that lipids are also sorted by vesicular traffic. Interactions between glycosphingolipids and apical proteins were postulated to aid the assembly of sphingolipid microdomains in the Golgi that would concentrate apical cargo as the first step in vesicle formation . This mechanism has two important features which distinguish it from the coat-mediated sorting:
• It also allows for the sorting of lipids.
• It works by actively excluding cargo that does not belong into the pathway and thus prevents the transported membrane from being diluted with inadequate material.
It has been shown previously, that basolateral proteins are excluded from the apical membrane , whereas the converse is not true [69, 117]. Physiologically this is sensible, since the apical membrane facing the lumen of an organ must be extremely resistant to external aggression by bile salt detergents, digestive enzymes or low pH, and its composition must therefore be tightly controlled. Whilst it is known that basolateral delivery depends on the interaction with adaptor proteins , domain-induced budding seems to be a mechanism ideally suited for delivery to the apical membrane.
Since these microdomains, or rafts, are believed to be small and dynamic, they must be clustered by proteins such as multivalent ligands or caveolin in order to be able to form a bud and later a vesicle or tubule. In apical raft delivery this has been postulated to be mediated by lectins or other multivalent cargo receptors [119, 120]. Raft and caveolar endocytosis is triggered by multivalent cargo, the best described being Simian virus 40 and cholera toxin [121-124], both of which bind several GM1 molecules . Here, the caveolar coat is not necessary for the membrane bending or vesicle formation, since rafts can endocytose upon clustering by a virus or toxin and be delivered to specific destinations in the cell without caveolin . In fact, the internalization has been shown to be faster in the absence of cav-eolin . Caveolin might thus not be necessary for the endocytic event as such, but rather add another level of regulation to this pathway, which is required for the efficient sorting of some ligands .
Indeed, caveolae membrane traffic does display special features that set it apart from other membrane traffic mechanisms. Caveolae were previously believed to be static structures , simply increasing the cell-surface area and keeping raft
Fig. 1.1 Two paradigms of cargo sorting and vesicle formation ► in membrane traffic: inclusion due to sorting signals followed by coat-driven budding (left), or exclusion due to phase separation and domain-induced budding (right). In the left column, proteins containing the appropriate cytoplasmic sorting signals (regardless of if they are residing in a raft or non-raft domain) are bound by adaptor proteins, on which the coat proteins assemble. For the clathrin-coat, membrane bending and subsequent budding is believed to be driven by a conformational change in the coat protein. In the right column, raft proteins are clustered by oligomerizing ligands or cytoplasmic scaffolding proteins, thereby excluding the group of non-raft proteins. Membrane bending and budding is driven by the need to minimize the line energy acting at the domain boundary.
1.8 Caveolae and Lipid Rafts in Membrane Traffic 15 Domain-induced budding
membrane available on the cell surface. Recently, it became evident that even in unstimulated fibroblasts and epithelial cells, 30% of the caveolae undergo local kiss-and-run cycles with the plasma membrane in which they pinch off and fuse again close to the original site . Upon receiving a trigger for endocytosis, caveolae switch from this short-range cycling to long-range cycling, resulting in an intermixing of cell-surface and intracellular caveolar vesicle pools and transport to caveosomes or endosomes . During the trafficking event, the caveolar coat seems to stabilize the clustered raft domain within the bilayer, so that it stays intact even after fusion with the acceptor compartment and can be re-used for multiple rounds of membrane trafficking . Cargo release at the target compartment must therefore also follow different principles than in the clathrin-coated vesicle traffic where the coat disassembles before fusion. Caveolae apparently keep their cargo sequestered, until its release is triggered by a compartment-specific cue. Cholera toxin is released upon encounter of a low pH environment in early endo-somes, but stays sequestered in caveolae in the neutral environment at the plasma membrane or in caveosomes. This type of membrane traffic seems especially suited for the sorting of non-membrane spanning cargo, in particular glycosphin-golipid-binding ligands .
The vesicle fusion machinery on the target compartment also has been proposed to be organized into domains of different lipid composition. The apical t-SNARE syntaxin 3 was proposed to reside in raft domains . More recent investigations have claimed that indeed different SNAREs are compartmentalized in the plasma membrane with the help of lipid domains, with syntaxin 3 residing in raft domains, syntaxin 2 being excluded from raft domains, and syntaxin 4 being equally distributed between the two . In polarized epithelial cells, syntaxin 4 resides on the basolateral surface, whereas syntaxin 2 and 3 are localized to the apical surface . The data would thus imply, that there could be two pathways trafficking to the apical side of epithelial cells - one raft- and one non-raft pathway. Indeed, it was previously observed that two different apical proteins, sucrase-iso-maltase and lactase-phlorizin-hydrolase, use separate containers for transport to the apical membrane of Madin-Darby canine kidney (MDCK) cells, and the existence of two different pathways was proposed .
Research on rafts and caveolae is entering a new phase. The technologies that have been used to study these membrane domains are being revised, and new technologies must be developed. If rafts are small and dynamic, many of the standard techniques that have been employed to visualize them (e.g., FRET, single particle tracking, FRAP in most cases) can not provide anything else but negative results because they are not suited for the size and time resolution needed. Another critical point is the purification methods used to isolate rafts or caveolae. The two were often confused with each other since they were supposed to co-fractionate when isolated based on detergent insolubility or light buoyant density. It is now accepted that these fractions are useful to obtain information about the proteins found in them, but since they form during the purification process, they can not be assumed to represent an equivalent of any pre-existing cellular domain, neither rafts nor caveolae . Instead, new approaches have been taken - for example, to isolate plasma membrane fragments with small antibody-coated beads . Techniques such as this must be developed in order to obtain pure raft and caveolae fractions that can be used to analyze their lipid and protein composition. With the new mass spectroscopic techniques it should then be possible to compare the lipidome of rafts and caveolae with each other to determine how similar they actually are, and also to compare them with the lipidome of the plasma membrane. Only then will we have a chance to assess properly the involvement of lipids in processes such as raft dynamics, raft clustering, and to address the special functions of caveolae.
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