And Lipid Protein Interactions

As suggested from an evolutionary perspective, proteins could have sensed in different ways the lateral structure imposed by the heterogeneous composition of the lipid matrix when interacting with the membrane. Partition of a protein from the aqueous bulk phase into the membrane interface requires the initial establishment of thermodynamically favorable interactions of specific protein regions with groups at the interfacial environment. Structural contributions to membrane partitioning, both at the sequence and at the conformational level, of membrane-associating proteins have been extensively analyzed in recent years (Wimley and White 1996; White and Wimley 1998). Once at the membrane interface, the properties of particular protein regions could define their eventual insertion or translocation into the most hydrophobic regions of the membrane. Lipid packing and lateral pressure are likely additional determinants influencing the interaction and insertion of proteins into the membrane (Marsh 1996; Tillman and Cascio 2003; van den Brink-van der Laan et al. 2004). Common experiments have used monolayer models for the determination of the maximal pressure permitting interaction of any given protein or peptide with a lipid layer. Only proteins increasing the surface pressure above 30 mN/m are typically considered as potentially competent to interact with deep regions of the membranes (Brockman 1999). Lateral heterogeneities in the membrane could regionally differ in properties such as: (1) the chemical interfacial environment that defines the initial partitioning of polypeptides from the bulk phase, (2) the transient exposure and accessibility of hydrophobic regions of the membrane, (3) the lipid packing and lateral pressure, or (4) the thickness of the membrane.

There are several examples in the literature of proteins that seem to show preferential interaction with defined membrane regions of a particular lipid compo sition or structure. It is difficult to evaluate what is the most prominent factor for such discrimination. Different membrane proteins have different membrane requirements for both initiating the lipid-protein interaction and stabilizing a

Fig.5.4. Lateral distribution of hydrophobic proteins SP-B and SP-C In monolayers and bi-layers of pulmonary surfactant. Lateral separation of ordered and disordered regions In pulmonary surfactant layers results In accumulation of proteins SP-B and SP-C In the most disordered phases In both interfaclal phospholipid films [left images) or giant vesicles formed with material from surfactant organic extract (SOE) (right images). Scale bars In left panels represent 25 pm and Images In right panels also have 25 pm width

Fig.5.4. Lateral distribution of hydrophobic proteins SP-B and SP-C In monolayers and bi-layers of pulmonary surfactant. Lateral separation of ordered and disordered regions In pulmonary surfactant layers results In accumulation of proteins SP-B and SP-C In the most disordered phases In both interfaclal phospholipid films [left images) or giant vesicles formed with material from surfactant organic extract (SOE) (right images). Scale bars In left panels represent 25 pm and Images In right panels also have 25 pm width given disposition. For example, the thickness of particular membrane patches has been proposed to be a major determinant for promoting proper protein sorting during intracellular trafficking (Harder 2003). Regulated post-translational modifications, such as acylation, seem to direct specific proteins to particular membrane domains (Mukherjee et al. 2003; Neumann-Giesen et al. 2004; Shogomori et al. 2005). Therefore, the lateral structure of the lipid matrix could impose major restrictions on the lateral distribution and sorting of the membrane proteome. Preferential distribution of any given membrane protein into a particular region or phase of the membrane presumably defines important functional features, such as local protein densities or the probability of the protein encountering homologous or heterologous counterparts (Fig. 5.4).

As a consequence, environmental conditions producing alterations in the lateral structure of the membrane could lead to profound rearrangements of membrane-associated protein distributions. This would have major implications on the establishment and organization of selective protein-protein interaction networks. It remains to be demonstrated whether cellular membranes actually support such dynamic behavior under physiologically relevant conditions. A recent study has demonstrated that the transfer of transmembrane protein segments into the membrane of the endoplasmic reticulum, as it passes through the translocon, is predominantly mediated by the thermodynamic rules governing lipid-protein interactions (Hessa et al. 2005). It still remains to be determined how the structure of the membrane itself, including the existence of compositional and structural heterogeneities, influences membrane protein assembly in vivo. It has been proposed that the composition of natural membranes may have been optimized to maintain the membranes close to the edge of an abrupt structural transition. This would have potentially major effects on the membrane lateral structure and domain organization (Grabitz et al. 2002). Membranes at that "edge" might show a very dynamic behavior that is sensitive to respond, perhaps via redistribution of the protein network, to environmental demands.

Membrane regions at the boundaries between different phases or domains could be especially amenable for supporting lipid-protein interactions. These boundaries have been proposed to posses lipid packing defects, which could be used by certain protein motifs to access deeper regions of the membrane interface (Saez-Cirion et al. 2002; Barlic et al. 2004; Cruz et al. 2004). Therefore, some proteins might require the presence of membrane heterogeneities to initiate partitioning into the membrane surface. Experiments with monolayers suggest that the boundaries between different phases, in systems showing liquid-expanded/ liquid-condensed phase coexistence, act as thermodynamic "sinks", trapping proteins or other molecules, virtual "impurities", that do not mix well with the lipid matrix (Fig. 5.5) (Ruano et al. 1998).

Some membrane-associated proteins may accumulate into these boundaries, where the dynamics may directly regulate important structural and functional protein features. It has been suggested that association of certain protein segments with the boundaries of some membrane domains could precede protein accumulation and membrane translocation (Saez-Cirion et al. 2002; Barlic et al. 2004).

Fig. 5.5. Examples observed in interfacial films of preferential association of proteins with the boundaries of condensed domains, (a) Formation of clusters of pretransmembrane peptide from HIV protein gp41 associated with raft-like domains of monolayers made from POPC/SM/ Choi (2/1/1, mole ratio) at 32 mN/m and 25°C. Green fluorescence is emitted by the probe fluo-rescein-dipalmitoylphosphatidylethanolamine (FL-DPPE) and red fluorescence from rhodamine-labeled peptide (Saez-Cirion et al. 2002). (b) Accumulation of the cytolysin equinatoxin-ll atthe boundaries between ordered and disordered phases in the monolayers ofSM/PC/Chol (50/14/35, mol ratios) compressed to 25 mN/m at 25°C. Green fluorescence was emitted from NBD-PC and red fluorescence from Texas Red-labeled protein (Barlic et al. 2004). (c) Accumulation of Texas Red-labeled surfactant protein SP-C in the surroundings of liquid-condensed domains of DPPC compressed to 11 mN/m. (d) Two-dimensional clusters of surfactant protein SP-B segregated to the boundary regions close to liquid-condensed domains of DPPC interfacial films observed byAFM (Cruzet al. 2004)

Fig. 5.5. Examples observed in interfacial films of preferential association of proteins with the boundaries of condensed domains, (a) Formation of clusters of pretransmembrane peptide from HIV protein gp41 associated with raft-like domains of monolayers made from POPC/SM/ Choi (2/1/1, mole ratio) at 32 mN/m and 25°C. Green fluorescence is emitted by the probe fluo-rescein-dipalmitoylphosphatidylethanolamine (FL-DPPE) and red fluorescence from rhodamine-labeled peptide (Saez-Cirion et al. 2002). (b) Accumulation of the cytolysin equinatoxin-ll atthe boundaries between ordered and disordered phases in the monolayers ofSM/PC/Chol (50/14/35, mol ratios) compressed to 25 mN/m at 25°C. Green fluorescence was emitted from NBD-PC and red fluorescence from Texas Red-labeled protein (Barlic et al. 2004). (c) Accumulation of Texas Red-labeled surfactant protein SP-C in the surroundings of liquid-condensed domains of DPPC compressed to 11 mN/m. (d) Two-dimensional clusters of surfactant protein SP-B segregated to the boundary regions close to liquid-condensed domains of DPPC interfacial films observed byAFM (Cruzet al. 2004)

A specialized case may exist for proteins possessing structural motifs with the ability to recognize specific lipid species, such as occurs with the increasing number of cholesterol-binding proteins (Shimada et al. 2002; Khan et al. 2003). Aromatic side-chains in these cholesterol-binding motifs seem to associate favorably with the planar cholesterol rings and form clusters that mutually potentiate both lipid and protein segregation. Interaction of these proteins with membranes would occur predominantly in cholesterol-enriched domains, where proteins with such motifs could accumulate.

It is debatable whether the selective interaction of certain membrane proteins with selected lipid species is sufficient to initiate organization of lipid domains in the plane of the membrane, which might consequently produce further lipid and protein sorting. It has been shown that the acetylcholine receptor accretes a phosphatidic acid-enriched membrane environment (Poveda et al. 2002). Likewise, some cholesterol-binding proteins have been proposed to nucleate formation of cholesterol segregates that, by virtue of selective cholesterol-sphingolipid interactions, could progress towards producing a raft-like membrane structure (Epand et al. 2001; Epand et al. 2003). Protein initiated reorganization of the lateral structure of the membrane could be the signal to recruit other lipid and protein partners. This has been proposed for the molecular mechanisms behind certain signaling platforms (Zhang et al. 1998; Bunnell et al. 2002). While the literature suggests apparently contradictory models confronting lipid-directed versus protein-directed mechanisms for organization of in-plane membrane structure, there is no doubt that an intense "lateral" cross-talk between lipids and proteins occurs and is responsible for the complex structure of real cellular membranes. The lateral cross-talk between lipids and proteins as potential regulatory mechanisms of cellular function is only starting to be envisaged.

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