Golgi sorting: Rafting to the top

Lipidomics Gateway (27 May 2009) [doi:10.1038/lipidmaps.2009.7]

New techniques for vesicle analysis implicate lipid rafts in sorting plasma membrane cargo at the trans-Golgi network.

Three-dimensional tomographic reconstruction of the Golgi complex, showing the cis-Golgi network (white) and the stack with the terminal three trans-cisternae in pink, red and cyan. The TGN (violet) appears as a tubular network that emerges from the lateral part of the last trans-cisterna (cyan).

Dynamic vesicular traffic within eukaryotic cells establishes and maintains differences in the lipid and protein content of membranes at particular organelles or the cell surface. Some aspects of the selection process of vesicular cargo are understood. However, much of the sorting mechanism, particularly of lipids, has been harder to clarify, in part because of difficulties with the reliable separation of vesicle populations and analysis of lipid species. Simons and colleagues have now devised a novel approach, taking advantage of recent advances in lipidomics, specifically to analyze post-Golgi secretory vesicles. Their work in the Journal of Cell Biology suggests that lipid rafts are key to the sorting of cargo at the trans-Golgi network (TGN).

Lipid rafts are dynamic membrane microdomains containing specific lipids and proteins. Their composition, enriched with sphingolipids and sterols, allows close packing of molecules, increasing membrane order and reducing fluidity. At least two lines of evidence suggest that raft formation is involved in cargo sorting: the concentration of raftophilic sterols and sphingolipids increases from the ER, through the Golgi to the plasma membrane, and mutations in yeast genes involved in biosynthesis of these lipids have trafficking defects.

To test whether TGN-derived secretory vesicles are indeed enriched in sterols and sphingolipids, the authors devised an isolation procedure. They engineered a bait protein, FusMidp, based on a chimera that is delivered in light-density secretory vesicles from the TGN to the plasma membrane in yeast. This Myc-tagged construct is glycosylated, allowing transit through the Golgi to be verified in isolates. A protease cleavage site was added to allow specific release of captured FusMidp-containing vesicles. After standard fractionation, the FusMidp vesicles were purified using antibodies and an immunoadsorbent surface: a mouse anti-Myc antibody initially pulled out the tagged vesicles and these were simultaneously depleted of contaminating endosomes using an antibody to an endosomal t-SNARE. The target vesicles were released from the immunoadsorbent surface by specific proteolytic cleavage. Their protein content, morphology and density were all consistent with the target population of TGN-derived light-density secretory vesicles. To validate this, the authors targeted a protein that cycles between the TGN and endosomes to isolate these pre-secretion compartments.

A novel quantitative lipidomics strategy recently described by this same group allows accurate lipidome-wide quantification of individual molecular lipid species using mass spectrometry. Quantifying 83 lipid species from 12 classes, the authors found that the FusMidp vesicles are enriched in yeast sterols and sphingolipids compared with the TGN and endosomes. They are also higher in phosphatidic acid and depleted in phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine.

Although this study does not address how raft clustering is initiated, it does support the idea that formation of lipid rafts is integral to sorting cargo at the TGN. The new approach can be applied to other compartments to help characterize all cellular traffic and hopefully illuminate the underlying mechanisms.

Emma Leah