Down-regulation of important transmembrane signaling receptors, including epidermal growth factor receptor (EGFR), is critical for cellular homeostasis by modulating the kinetics and amplitude of signaling cascades. Following endocytosis, activated signaling receptors are sorted through the endosomal compartment. A subset of late endosomes facilitate receptor trafficking into intralumenal vesicles (ILVs) within the endosomal lumen by a mechanism known as multivesicular body (MVB) sorting. Receptors that enter the MVB sorting pathway are degraded following endo-lysosomal fusion events. ILV formation requires membrane budding with a topology away from the cytoplasm, a phenomenon that is mediated by the endosomal sorting complexes required for transport- III (ESCRT-III), and the AAA-ATPase, Vps4. Vps4 ATPase activity is regulated by interactions with some ESCRT-III subunits, and mutations that are predicted to disrupt this process are implicated in neurodegenerative disease. Some ESCRT-III subunits, including Vps2 and Did2, stimulate Vps4 ATPase activity using MIT-interacting motif 1 (MIM1). One ESCRT-III subunit, Ist1, inhibits Vps4 activity in vitro, although the mechanisms underlying this regulation and its roles in MVB sorting are unclear. In addition, we have demonstrated that Ist1 is also able to stimulate Vps4 activity in vitro in a MIM1- MIT-dependent manner under elevated salt conditions. This finding may be related to an in vivo mechanism that converts Ist1 from an inhibitor to stimulator of Vps4 activity following conformational changes from a "closed" to "open" state that occur during ESCRT-III polymerization. Ist1 is incorporated into ESCRT-III polymers by Did2, suggesting that interactions with Did2 represent the physiologically relevant mode of Ist1 conversion to the "open" conformation in vivo. The goals for this research training program are to utilize yeast genetic, cellular biological, and biochemical approaches to (1) elucidate the molecular mechanisms underlying distinct modes of Vps4 regulation by Ist1 and (2) determine the roles of the Ist1 inhibitory and stimulatory domains in mediating MVB sorting in vivo. Completion of this work will provide an increased understanding of how Vps4 activity is coordinated by multiple ESCRT-III subunits leading to MVB sorting, a critical life process that is implicated in neurodegenerative disease. The insights gained from this research will have broad application to the study potential mechanisms underlying ESCRT-III-mediated regulation of other AAA- ATPases, such as Spastin, which interact with Ist1 and are also implicated in neurodegenerative disease.
Defects in degradative protein trafficking pathways, including multivesicular body (MVB) sorting, are implicated in cancer and neurodegenerative disease. Vps4 is an essential enzyme in MVB sorting that is regulated by endosomal sorting complex required for transport-III (ESCRT-III). This project aims to elucidate the molecular mechanisms underlying Vps4 regulation by Ist1, a unique ESCRT-III subunit that is able to both inhibit and stimulate Vps4 activity, and to determine its roles in facilitating MVB sorting.
|Davies, Brian A; Norgan, Andrew P; Payne, Johanna A et al. (2014) Vps4 stimulatory element of the cofactor Vta1 contacts the ATPase Vps4 ?7 and ?9 to stimulate ATP hydrolysis. J Biol Chem 289:28707-18|