Spatiotemporal dynamics and function of multisubunit trafficking complexes with dual functionality within the endolysosomal pathway

几条证据表明，溶酶体/空泡HOPS和内体CORVET复合物的动态性远远超过从凝胶上纯化的六聚体复合物的概况中预期的。在酵母中，可能在更高的真核细胞，内体似乎积累在不同的网站接近液泡，这可以通过过度表达的CORVET亚基Vps 8增强。同样地，Vps 39在HOPS之外具有至少两个额外的功能，既在vCLAMP形成中又在调节TORC 1中。这些Rab-specific亚基是如何从它们的六聚体中分解的，以及这是如何被调节和控制的，在很大程度上还没有被探索。在这个项目中，我们将剖析HOPS和CORVET如何组装和拆卸，以释放在其他交通相关过程中发挥作用的单个亚基，这些过程超出了束缚和融合，无论是在酵母中，还是后来在后生动物细胞中。在目标1中，我们将揭示的Rab-specific亚基的CORVET和HOPS的动态，重点是它们的分布，复杂的组装和拆卸的调节，以及功能超越拴系复合物。特别是，我们将解决Vps 39功能在TORC 1的调节。目的2将集中在HOPS Vps 41和CORVET Vps 3的混合复合物的作用，在那里它的形成，定位，功能和生理学将被澄清。在目标3中，通过监测Vps 41和Vps 39相对于已知Rabs和外壳蛋白（如AP-3）的动态和调节，将对HOPS（以及后来的CORVET）动态的见解转移到后生动物细胞中。因此，我们将揭示新的见解，在很大程度上未开发的装配和拆卸的HOPS和CORVET及其作用的内溶酶体功能。

Several lines of evidence indicate that the lysosomal/vacuolar HOPS and endosomal CORVET complexes are far more dynamic than anticipated from the profile of the purified hexameric complexes on gels. In yeast, and possibly in higher eukaryots, endosomes seem to accumulate at distinct sites proximal to the vacuole, which can be enhanced by overexpression of the CORVET subunit Vps8. Likewise, Vps39 has at least two additional functions outside of HOPS, both in vCLAMP formation and in regulating the TORC1. How these Rab-specific subunits disassemble from their hexamers and how this is regulated and controlled remains largely unexplored. Within this project, we will dissect how HOPS and CORVET assemble and disassemble to release individual subunits that function in other trafficking-related processes beyond tethering and fusion, both in yeast, and later in metazoan cells. In Aim 1, we will reveal the dynamics of the Rab-specific subunits of CORVET and HOPS with a focus on their distribution, regulation of complex assembly and disassembly, and functions beyond the tethering complexes. In particular, we will address the regulation of the Vps39 function at TORC1. Aim 2 will focus on the role of the hybrid complex of HOPS Vps41 and CORVET Vps3, where its formation, localization, function, and physiology will be clarified. In Aim 3, insights on HOPS (and later CORVET) dynamics will be transferred into metazoan cells by monitoring dynamics and regulation of Vps41 and Vps39 relative to known Rabs and coat proteins such as AP-3. We will thus reveal novel insights into the largely unexplored assembly and disassembly of HOPS and CORVET and its role in endolysosomal function.