New Insights Into The Transport Machinery Of The Cell: A Single Protein Substitutes The Regulatory Machinery Specifying Endosome Fusion In Vitro

February 18, 1999

Endosomes are a good system to study how cells organise their internal transport logistics. Cells constantly internalise material from the external of the cell and distribute it to the other cellular compartments via specific organelles called endosomes. To bridge the distance between the cell surface and the endosomes, small membrane engulfed vesicles bud from the cell surface like soap bubbles and surround the cargo. Inside the cells there are thousands of such shuttle vesicles that have an important task: to specifically find their target organelle, dock with it and deliver their cargo by fusing with the target membrane. These organelles are surrounded by cytosol, the aqueous solution making up the inside of the cell and through which the vesicles navigate. The complexity comes from the fact that cytosol contains thousands of molecules and for a long time only a few have been recognised to regulate the specificity of the docking and fusing process. Researchers from the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden/Germany, the European Molecular Biology Laboratory (EMBL) and collaborators from the University of Liverpool now successfully used purification and in vitro experiments to analyse the cytosolic proteins essential for this complex biological mechanism. They reported the new findings in this week's issue of "Nature" (Nature, Vol. 397, 18 February 1999).

In a first step it was necessary to separate proteins, which are involved in the regulation of endosome fusion from those, which are not. Starting point of this strategy was a family of proteins called Rabs, which are established key players in the activation of vesicle docking and fusion. Rabs act as molecular switches alternating between two stages: when active, they allow vesicle membranes to dock and fuse their targets, in their inactive state, fusion doesn't occur. "Rab5" is known to be a key player responsible for fusion of vesicles coming from the cell surface with endosomes. But it was known that Rab5 does not act alone: effectors are required for membrane fusion activity. Nevertheless, the view of the machinery regulating this process was far from being complete. So in a first step the scientists searched for all proteins binding to Rab5 and therefore were presumably involved in regulating this process.

A specific protocol (affinity chromatography) was designed for this purpose and out of all cytosolic proteins it led to the purification of 22 proteins associated with Rab5. In a next step it was demonstrated that a mix of these 22 proteins was able to functionally substitute cytosol in an experimental strategy which monitors early endosome fusion in an in vitro assay. In order to further investigate which of the proteins form the minimum machinery in this process, different protein combinations were tested. Most surprisingly it was found that a single protein, called EEA1, was necessary and sufficient to confer minimal fusion activity. Other experiments suggest that Rab5 and other associated proteins acted upstream of EEA1, implying, that these Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. This regulatory machinery is important in vivo but can be bypassed by the increase of EEA1 concentration in the minimalist in vitro system. It was found that EEA1 actually bridges membranes and this knowledge now changes our view of endosome fusion specificity.

It is a long postulated hypothesis that the pairing of two corresponding proteins called vSNARE and tSNARE - which fit together like a key and a lock - is the primary molecular event specifying vesicle targeting and fusion. The new data now make clear that EEA1 acts before the SNAREs. EEA1 is thought to behave as a kind of docking molecule, which allows the SNARE-key/lock to pair and together with the SNAREs regulates the specificity of early endosome fusion.

The protocol used for the identification of the Rab5 regulated fusion machinery is a perfect model system to study the other Rab-dependent pathways in internal cell transport. After a more detailed characterisation of the molecular players of the transport route from the cell surface to the endosomes, the same system will be extended to investigate the transport leaving endosomes: the mechanism through which the internalised material is further distributed throughout the cell as well as the transport circuit which recycles membrane vesicles back to the plasma membrane. By this strategy it is hoped to identify and understand a central biological mechanism to an extent so far rarely known.


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