Intracellular membrane trafficking fundamentally involves vesicles that bud from various membranes to be transported elsewhere within the endomembrane system of the eukaryotic cell. Our lab characterized the endosomal sorting complexes required for transport (ESCRTs) that induce a membrane budding process at the endosome that results in the formation of intralumenal vesicles (ILVs) inside the endosome, multivesicular endosome (MVE). This is a topologically inverted process from the formation of clathrin-coated endocytic vesicles, or COP-I and COP-II coated vesicles between the endoplasmic reticulum and the Golgi complex. A number of seemingly unrelated biological processes, including intralumenal vesicle formation inside multivesicular endosomes, enveloped virus budding at the plasma membrane (e.g., HIV), and the abscission event of metazoan cytokinesis, require the ESCRT machinery.
The ESCRTs comprise a pathway of five distinct complexes – ESCRTs-0, -I,-II, -III, and Vps4. Our long-term goal is to understand the molecular mechanisms that underlie the assembly and function of each of the ESCRT complexes in the formation of multivesicular endosomes for the delivery of cargo destined for degradation in the vacuole or lysosome. Previous studies in the lab set the framework establishing the ESCRT complexes as ubiquitin binding, cargo clustering and sequestering, and membrane remodeling machinery. Thus, the ESCRT pathway can be viewed as a cargo recognition and membrane deformation machine that can be analyzed from three distinct perspectives: 1) the membrane they deform, 2) the ESCRT proteins themselves, and 3) the cargoes they sort.
ESCRT-III has been shown to be the minimal machinery required for cargo sequestering and membrane invagination. Our current research interests focus on dissecting the protein-protein and protein-membrane interactions associated with the ESCRT-III complex. Using genetics, we found that ESCRT-III assembles transiently and dynamically on the endosomal membranes in a sequential order: Vps20-Snf7-Vps24-Vps2. The ESCRT-III complex is subsequently disassembled by the recruitment of the final ESCRT complex, the AAA-ATPase Vps4. By in vitro reconstitution assays using purified ESCRT-III components and model lipid systems, we described Snf7 filaments of ~9 nm that are converted into spiraling protein helices with the addition of Vps24 and Vps2. When coassembled with ESCRT-II, ESCRT-III forms ~65 nm diameter rings, indicative of a cargo-sequestering supercomplex. We have also recently identified two essential modules for ESCRT-III-membrane association. Our results indicate that ESCRT-III is tuned to maintain the topological constraints associated with protein filament-mediated membrane invagination and vesicle formation. Present work in the lab focuses on further investigating the conformational dynamics and molecular architecture of the ESCRT-III complex, through both in vivo genetic screening, and in vitro biochemical techniques.
Key Publications
1. D. J. Katzmann, M. Babst, S. D. Emr (2001). Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell. 106, 145–155 .
2. W. M. Henne, N. J. Buchkovich, S. D. Emr (2011). The ESCRT pathway. Dev Cell. 21, 77–91.
3. W. M. Henne, N. J. Buchkovich, Y. Zhao, S. D. Emr (2012). The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices. Cell. 151, 356–371.
4. S. Tang, W. M. Henne, P. P. Borbat, N. J. Buchkovich, J. H. Freed, Y. Mao, J. C. Fromme, S. D. Emr (2015). Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments. elife. 4, doi:10.7554/eLife.12548.
5. S. Banjade, S. Tang, Y. H. Shah, S. D. Emr (2019). Electrostatic lateral interactions drive ESCRT-III heteropolymer assembly. Elife. 8.
