| Description |
The multivesicular body (MVB) is a late endosomal compartment containing intralumenal vesicles enriched with a subset of transmembrane cargoes that form as a result of the inward budding of the endosomal limiting membrane. In Sacchoromyces cerevisiae ESCRT (endosomal sorting complexes required for transport) machinery, consisting of distinct protein complexes ESCRT-0, I, II and III, together with the AAA+ (ATPases associate with the variety of cellular activities) Vps4 (vacuolar protein sorting 4) ATPase are responsible for the MVB sorting. Solubilization of ESCRTs by the active Vps4 oligomer is thought to be the final step in the biogenesis of MVB. Vps4 consists of N-terminal ESCRT-III-interacting MIT (microtubule interaction and trafficking) domain, C-terminal nucleotide ATPase domain and the linker region that connects them. Function of the Vps4 is regulated through its recruitment from the cytoplasm through a complex network of interactions with ESCRT-III-associtaed proteins Did2, Ist1, Vta1 and Vps60 to the endosomeassociated ESCRT-III consisting of Vps20, Snf7, Vps2 and Vps24 where Vps4 assembles into an active oligomer. ATPase domains of Vps4 subunits promote oligomerization into closed rings and two stacked rings form an active Vps4 oligomer that contains a functionally important central cavity. Position of the MIT domains in the oligomer varies, suggesting that function of the active Vps4 oligomer might require flexibility in movement of the MIT domains. In Chapter 2, we have perfomed a detailed in vivo analysis of the interactions that mediate recruitment of Vps4 to ESCRT-III. Our data revealed a high degree of redundancy in the Vps4 interaction network. In our model, we propose that interactions with Did2 and Ist1 recruit Vps4 from cytoplasm to ESCRT-III and subsequent interaction with Vta1 promotes formation of an active Vps4 ATPase. We speculate that two rings that comprise active Vps4 oligomer have different functions - one ring is important for the ATP hydrolysis and substrate processing by Vps4, whereas the second ring is not directly involved in the ATP hydrolysis and serves a regulatory role. In Chapter 3, we have investigated if MIT domains need to be tucked away from the central cavity of the active Vps4 oligomer to allow processing of a substrate. We performed detailed functional and biochemical analysis of the serial deletions within the linker region. In our model, we propose that MIT domains are positioned around the central cavity region and cooperate together in solubilization of a substrate. |
