Determining the roles of Ist1 inhibition and stimulation of Vps4 ATPase activity

描述（由申请人提供）：下调重要的跨膜信号受体，包括表皮生长因子受体（EGFR），通过调节信号级联的动力学和幅度对细胞稳态至关重要。内吞作用后，激活的信号受体通过内体隔室进行分类。晚期核内体的一个子集通过一种被称为多泡体（MVB）分选的机制促进受体转运到核内体腔内的腔内囊泡（ILVs）中。进入MVB分选途径的受体在内溶酶体融合事件后被降解。ILV的形成需要远离细胞质的膜出芽，这一现象是由运输所需的内体分选复合物-III （ESCRT-III）和aaa - atp酶Vps4介导的。Vps4 atp酶活性是通过与一些ESCRT-III亚基的相互作用来调节的，预计会破坏这一过程的突变与神经退行性疾病有关。一些ESCRT-III亚基，包括Vps2和Did2，通过mit相互作用基序1 （MIM1）刺激Vps4 atp酶活性。一个ESCRT-III亚基Ist1在体外抑制Vps4活性，尽管这种调控的机制及其在MVB分选中的作用尚不清楚。此外，我们已经证明，Ist1也能够刺激Vps4的活性

DESCRIPTION (provided by applicant): 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.