The building block of living things is the cell, and every cell has specific compartments with dedicated functions, akin to the separate rooms in a house. Therefore, to construct a cell, its components, especially proteins and lipids that constitute the membrane barriers surrounding each compartment, must be accurately sorted to, and properly assembled at, the correct destination and at the right time. The landmark discoveries made by this year’s Shaw Laureate in Life Science and Medicine, Scott D Emr, provided ground-breaking insights into the composition, dynamics, and assembly of a specialized subset of these membrane-enclosed compartments.
Among the compartments with discrete functions are organelles, like the lysosome in animal cells and its equivalent in yeast cells, the vacuole. One mechanism for transport of material to such a destination is through delivery via vesicles, small, fluid-filled and protein-containing sacs surrounded by a lipid membrane. In pioneering studies, Emr devised an elegant genetic strategy to identify genes in budding yeast, a model cell, that encode components required to build the vacuole. Emr fused the gene encoding the lysosomal protease carboxypeptidase Y (CPY) to the gene encoding invertase (INV), an enzyme normally secreted to permit growth of this yeast on the sugar sucrose. As anticipated, the CPY-INV fusion protein was transported efficiently into the vacuole and not secreted, indicating that the organelle-targeting information in CPY was dominant. Mutant cells unable to deliver CPY-INV properly, causing the fusion protein to be secreted, could be identified by their ability to grow on sucrose medium. Such vps mutants (defective in vacuolar protein sorting) secreted every other vacuolar component tested, indicating that VPS gene products had general roles in vacuole biogenesis. More than 40 VPS genes were identified and conserved counterparts found in human cells. Emr began the arduous, but immensely important, task of determining the biochemical functions of the Vps proteins. Indeed, understanding the role of each Vps protein would have a transformative impact on our knowledge about the molecular basis of intracellular vesicle-mediated protein trafficking.
A subset of VPS genes revealed a key role for phosphoinositide (PI) lipids as organelle “identity tags” that direct vesicle trafficking. The VPS34 gene specifies a lipid kinase that phosphorylates PI to generate PI-3P on the surface of a pre-lysosomal compartment, the endosome. This key finding led the way for showing that other phosphoinositides act as “address codes” for other cellular membranes. Another large subset of VPS genes was found to encode components of an ordered pathway that brings about dramatic membrane deformation and scission, which Emr named the ESCRT (Endosomal Sorting Complexes Required for Transport) machinery. In a series of truly path-finding discoveries, Emr and colleagues systematically characterized these components, defining five distinct stages in execution of the ESCRT pathway and identifying the proteins and protein complexes that function at each step: ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, and the Vps4 complex (an AAA+ ATPase). The critical requirement for PI-3P was pinpointed by showing that ESCRT-0 and ESCRT-II are selectively recruited to the endosomal membrane by binding to this lipid. Moreover, ESCRT-0, ESCRT-I and ESCRT-II were shown to bind ubiquitin, a “tag” specifically attached to membrane proteins destined for packaging into ESCRT-generated vesicles. As a result, ESCRT-0, ESCRT-I, and ESCRT-II are recruited in succession and co-assemble on the endosome surface, where they nucleate co-recruitment of the ESCRT-III components (Vps20, Vps32, Vps24 and Vps2). The Emr group then made the remarkable discovery that the ESCRT-III components polymerize into a spiral of filaments that corral cargo captured by the other ESCRTs and, most dramatically, sculpt the membrane, bending it away from the cytoplasm into the endosome, thereby depositing the bound cargo into invaginations projecting into the endosome. The Vps4 ATPase catalyzes resolution of the process, driving scission of the membrane at the necks of the invaginations, culminating in release of vesicles into the endosomal lumen and in disassembly and recycling of the ESCRT machinery into the cytoplasm. These seminal discoveries about how a membrane can be bent away from the cytoplasm established a new paradigm in membrane topology and shattered the previous dogma, in which vesicles formed for secretory transport and for clathrin-mediated endocytosis bend toward the cytoplasm. Emr’s pioneering research showed that failure of the ESCRTs to sort cargo into endosomes results in persistence of proteins (e.g., cell-surface receptors) on the endosome surface and their inappropriate recycling back to the plasma membrane, thus revealing how mutations in ESCRT components can contribute to cancers by permitting sustained signaling by growth factor receptors. ESCRT-directed membrane bending is now recognized as a universal mechanism used by cells in other ways, such as repair of membrane damage, completion of cytokinesis, and pruning of neuronal axons during brain development, as well as exploited by viruses, such as HIV, to bud and escape from host cells. Thus, Emr’s remarkable contributions have uniquely illuminated processes that are central for life, from yeast to humans .
28 October 2021 Hong Kong