The sequential action of enzymes has been shown to modify members of a class of membrane lipid called phosphoinositides to direct integral membrane proteins for recycling. See Letter p.408
The ability of cells to take up nutrients or receive signals from their neighbours depends on proteins that are integrated into the plasma membrane. These proteins are not static, and can be moved to the cell's interior through a process called endocytosis. Some will subsequently be routed back to the cell surface and reused, and others will be degraded. There are grave consequences if the balance between recycling and degradation is derailed. In this issue, Ketel et al.1 (page 408) define a molecular mechanism that underpins the decision to direct cargo for recycling.
Phosphatidylinositol (PI), a major class of phospholipid, is an integral component of cell membranes. Phosphorylation at one or more of three different positions (dubbed 3, 4 and 5) in the headgroup of PI, which protrudes from the membrane, can give rise to seven minor lipid species called phosphoinositides (PIPs). There is considerable evidence2 to suggest that PIPs are crucial intracellular traffic controllers. Different species of PIP recruit specific effector proteins, many of which direct the routing of membranes and their cargoes within the cell.
PI3P (a PIP marked by one phosphate group at position 3) is needed for the formation of membranous structures called early endosomes3, which contain endocytosed proteins. As early endosomes mature, their cargoes are sorted according to whether they will be recycled or degraded. Addition of a phosphate group to PI3P to form PI(3,5)P2 directs proteins for degradation4. However, it has not been known which PIPs, if any, mark membranes for recycling.
A PIP phosphatase enzyme called MTM1 removes the phosphate from position 3 of PI3P and PI(3,5)P2 (ref. 5). Mutations in MTM1 cause a severe, debilitating muscular disease in humans called X-linked centronuclear myopathy5. Ketel et al. analysed connective-tissue cells called fibroblasts taken from patients with X-linked centronuclear myopathy, and a cultured cell line in which MTM1 was downregulated by genetic manipulations. They observed that transferrin-receptor proteins, which are responsible for iron uptake and are normally recycled after endocytosis, became trapped in endosomes, unable to fuse with the plasma membrane in these cells.
Another protein that is normally recycled, β1 integrin, also became trapped in endosomes. This observation is in agreement with a previous study, in which the mtm gene was mutated in flies6, and is interesting because the presence of β1 integrin at the cell surface is crucial for many cell types, including muscle cells, to attach to their surrounding matrix7. Ketel et al. showed that it is the inability of the mutant MTM1 to remove phosphate from endosomal PI3P that prevents proper recycling of cell-surface molecules to the plasma membrane. This inability may contribute to the muscle defects seen in patients.
Next, Ketel et al. showed that, in parallel with the removal of phosphate from PI3P, a phosphate group is added to position 4 by a lipid kinase enzyme called PI4K2A, generating PI4P. The authors found that MTM1 and PI4K2A form a complex, and that PI4K2A is required for the recruitment of MTM1 to endosomes. PI4K2A and MTM1 also form a complex with the exocyst, a set of proteins that is essential for the fusion of recycling endosomes with the plasma membrane8. Thus, the authors have mapped a series of biochemical steps in membrane-protein recycling (Fig. 1) — beginning with the removal from the endosomal membrane of PI3P and its replacement with PI4P, and followed by the recruitment of various proteins — that prepare endosomes and their cargo proteins for return to the plasma membrane.
These results raise questions about the organization of the sorting process. Previous studies have suggested that PI4K2A is required for the degradation of some proteins (other receptor proteins such as epidermal growth factor receptors9, for example) and foreign particles ingested by immune cells called phagocytes10. PI4P is also found in late endosomes, which are intermediates for the degradation pathway11. The ability to discriminate between the same PI4P signals in membrane compartments destined for different fates probably involves extra molecular regulators.
It has previously been postulated that PIP effectors function by recognizing additional membrane components, such as the Rab proteins12. Rab11 is known to mark membranes destined for recycling, and Rab7 marks them for degradation12. These proteins, together with PI4P, might recruit different molecular machines that determine different membrane fates. Similar processes are seen in other PIP-regulated pathways13, such as membrane-curvature sensing or the transport of certain lipids between the membranes of different organelles, and is likely to be replicated in other lipid–protein complexes at different stages of endocytosis and recycling.
Ketel and colleagues' study marks a step forward in our understanding of how PIPs act as a 'lipid code' to direct the sorting of cell-surface molecules in endosomes. Moreover, they provide a neat demonstration of the importance of this code to preventing diseases such as X-linked centronuclear myopathy. Getting a grip on the molecular underpinnings of this debilitating disease may help scientists to devise strategies to improve its outcomes.Footnote 1
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