There are many examples of proteins that have been found in TX-DRMs without the use of proteomics, including various proteins anchored by glycosylphosphatidylinositol (GPI) in the exoplasmic leaflet of the membrane, and acylated intracellular protein tyrosine kinases of the Src family [21
]. Both these kinds of protein carry largely saturated, unbranched lipid modifications that would easily partition into a liquid-ordered domain. Recently, several groups have taken a proteomic approach to identify proteins in DRMs [11
]. Using the detergent Brij-58 to prepare DRMs, Bini et al
] found that 11 of 17 proteins identified by mass spectrometry were of mitochondrial origin, and plasma-membrane lipid-raft proteins were detectable only by western blotting, suggesting that the latter were of very low abundance in the Brij-58 DRMs. As mitochondrial membranes are not expected to contain lipid rafts and Brij-58 is a poor detergent for making DRMs enriched in lipid raft proteins, the relationship between the identified markers [16
] and lipid rafts is unclear.
In another study [12
], a subset of heavier TX-DRMs - those with a higher protein-to-lipid ratio - was found to contain numerous proteins of the membrane cytoskeleton [12
]. This result emphasizes the link between lipid rafts and intracellular structures, which has been touched on by several other studies reporting an enrichment of cytoskeletal proteins in DRMs (see, for example, [27
]). The link is not surprising, however, because Triton X-100 insolubility was originally a method for preparing the cytoskeleton.
Given the difficulty in solubilizing hydrophobic proteins for two-dimensional electrophoresis, various tricks have been used to identify proteins of low abundance and/or high hydrophobicity. Often these tricks lead to identification on the basis of only one peptide per protein, which does not always allow unambiguous identification by mass spectroscopy, but in most cases proteins can be successfully identified. Using a cysteine-specific biotinylation agent in combination with in-gel digestion, von Haller et al
] identified 70 proteins from Jurkat T-cell TX-DRMs. These could mainly be grouped into signaling and cytoskeletal proteins. This study [13
] also identified some proteins from cellular locations not expected to contain lipid rafts, but there have been an increasing number of reports of 'moonlighting' proteins that have different functions at different locations, so it is advisable to keep an open mind when using protein location to assess preparation purity.
The most recent proteomic study of DRMs is that of Matthias Mann and co-workers [14
]. This work is technically impressive: the ratios of isotopes from cells labeled with either leucine or trideuterated leucine were measured by mass spectrometry and used to group TX-DRM proteins into three categories on the basis of the sensitivity of their presence in the TX-DRMs to acute cholesterol depletion: raft proteins, raft-associated proteins and 'nonspecific' proteins. The cell type used (HeLa cells) contains caveolae as well as lipid rafts, and the methods used for lipid-raft purification do not distinguish between the two. The paper [14
] makes the assumption that the association of genuine raft proteins with TX-DRMs should be sensitive to cholesterol depletion whereas that of contaminating proteins should not, but this has not been universally established. Although cholesterol depletion does make some components of lipid rafts sensitive to detergent extraction, both lipid and protein markers of lipid rafts can still be purified in TX-DRMs after such treatment [28
]. There is also evidence that cholesterol depletion causes coalescence rather than dispersion of lipid domains in living cells [29
]. Mann and co-workers [14
] treated cells with methyl-β-cyclodextrin until no less than 96% of the cells' cholesterol had been removed. During this one-hour treatment the cells almost certainly lose viability, probably undergo extensive intracellular reorganization and degradation, and lose many proteins through membrane blebbing. The authors found that disorganization of the cholesterol in rafts using the agents nystatin and filipin did not identify any specific grouping of proteins sensitive to this, and nor did any of the three cholesterol-disrupting agents give rise to a useful discrimination when applied to floating membranes prepared in the absence of detergent, reinforcing the questionable utility of non-detergent methods for preparing 'lipid rafts'.
The supplementary information (Tables 3-5) to the study by Mann and colleagues [30
] gives complete and informative data on the proteins they identified. The 'nonspecific' category, showing low sensitivity to cholesterol depletion, reassuringly contains the transferrin receptor, the classical non-raft marker, as well as many other proteins not expected to be in rafts. In general, the authors find the expected proteins such as small and heterotrimeric G-proteins, Src-family tyrosine kinases and cytoskeletal proteins in the 'raft' category, with the addition of several glycolytic enzymes, ribosomal proteins and nuclear proteins. These non-traditional raft proteins may be examples of proteins with multiple functions; they do not necessarily reflect contamination. It is less straightforward to know where to draw the line between 'raft' proteins (defined as showing high dependence on cholesterol for association with DRMs) and 'raft-associated' proteins (showing intermediate dependence on cholesterol for DRM association). The cut-off values are set somewhat arbitrarily where there are minor discontinuities in the graph of proteins plotted in order of their dependence on cholesterol (see Figure 3 of Foster et al
]). This results in the classical caveolar or lipid-raft protein caveolin-1 being classified as only 'raft-associated' whereas by all other criteria this is a highly raft-enriched protein. In fact, caveolin-1 is very close to the cut-off between raft-associated and nonspecific proteins. The failure of caveolin-1 to show strong dependence on cholesterol for DRM association could be because the protein can itself bind cholesterol, perhaps making it less susceptible to the general loss of cholesterol from cell membranes, and this could also apply to other cholesterol-binding proteins.
Mann and co-workers [14
] conclude that lipid rafts are rich in signaling molecules, membrane-skeletal and cytoskeletal proteins, consistent with studies showing that lipid-raft components can associate with actin filaments, either directly or through adaptors, temporarily anchoring them to intracellular structures. Many of the proteins identified have not previously been reported to partition to lipid rafts, and they extend the list of identified raft proteins to 241. This is an impressive number given that TX-DRMs are estimated to contain only 0.3-2% of total cellular protein. Mann and co-workers [14
] also characterize a large number of hypothetical raft or raft-associated proteins, which may turn out to be the most useful aspect of this study because it points to new raft proteins as well as new raft functions. No doubt we will learn more about the organization of the plasma membrane once these proteins have been functionally assigned; the raft and raft-associated proteins identified so far suggest an involvement of lipid rafts in ubiquitinylation and endocytosis.
It is clear that the dynamic plasma membrane of cells is far from homogenous, and we are still only just scraping the surface of its complexity. With careful isolation of lipid rafts, proteomics may be a useful tool for understanding this complexity.