title: Dynamics and Architecture of the HOPS Tethering Complex in Yeast Vacuole Fusion
creator: Ostrowicz, Clemens Werner
subject: ddc-570
subject: 570 Life sciences
description: The evolvement of a complex endomembrane system, which is separating a variety of biochemical processes into distinct compartments, is a hallmark of eukaryotic cell development. Cellular homeostasis depends on the abilities of these lipid bilayer-enclosed organelles both to maintain distinct characteristics and to exchange materials. This is mainly achieved by a process called vesicular transport, which allows for a constant exchange of proteins, lipids and metabolites between different compartments. Lipid bilayer enclosed vesicles bud from the donor compartment, are transported to the target compartment and fuse with its surrounding membrane. The basic machineries involved in the process in budding and fusion have been intensely investigated in the last years. However, our knowledge about the processes, which confer target specificity and regulate intracellular membrane fusion, is still limited. Before fusion of two-compartments can occur, they have to specifically recognize and bind each other to allow for subsequent SNARE-induced fusion to take place. This early step in the fusion reaction is called tethering and involves the action of tethering factors and Rab GTPases. In my research, I focused on the HOPS protein complex that is implicated to function in the tethering process at the yeast vacuole, the fungal equivalent of lysosomes. To investigate the molecular properties that confer the functionality of this large hexameric complex, I established a method that allowed for the purification of substantial amounts of HOPS and investigated the interactions taking place between different subunits. This work paved the ground for electron microscopy analysis of the whole complex, which is currently performed and which yielded first, preliminary data. Furthermore, it allowed for the identification of the novel CORVET tethering complex at the endosome, which has several subunits in common with the HOPS complex. I was able to show that chimeric complexes exist, harboring both HOPS- and CORVET-specific subunits. This finding suggests that both complexes are dynamic and can interconvert. During my studies on the subunits’ interactions, I identified stable subcomplexes of the HOPS complex, for one of which I could show that it exists in vivo. The existence of such subcomplexes implies a much more dynamic functioning of the HOPS subunits than previously anticipated. This notion is further strengthened by my studies on the functionality of different subunits and subcomplexes. Intriguingly, my results show that the Rab Guanyl nucleotide exchange factor Vam6, which is needed to activate the vacuolar Rab Ypt7 for subsequent fusion and which is a component of the HOPS complex, loses its ability to interact with Ypt7 upon incorporation into a subcomplex or the fully assembled HOPS complex. In contrast to this, the subunit Vps41, which I could identify as a Rab effector, is active as a single protein and as part of the complex, suggesting that it might sequentially recruit the subcomplexes to assemble into the holo-complex at sites harboring active Ypt7. Another feature of Vps41 was addressed in my work. This protein was previously shown to be phosphorylated by the vacuolar casein kinase Yck3. I identified the phosphorylation sites in the Vps41 sequence, which allowed further studies on the effect of the phosphorylation on the functionality of the protein. In the phosphorylated state, the protein is displaced into the cytosol whereas it accumulates at endosomal-vacuolar fusion sites if phosphorylation is prevented. Intriguingly, we found that Ypt7 overexpression is able to partially rescue the loss of localization in the phosphomimetic mutant, indicating a cross-talk between these two layers of Vps41 regulation.
date: 2009
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/9655/1/Ostrowicz.pdf
identifier: DOI:10.11588/heidok.00009655
identifier: urn:nbn:de:bsz:16-opus-96554
identifier:   Ostrowicz, Clemens Werner  (2009) Dynamics and Architecture of the HOPS Tethering Complex in Yeast Vacuole Fusion.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/9655/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng