Browsing by Author "Barbosa, Sonia Cristina de Oliveira"

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Identification and characterization of Dop1p as an essential component of the Neo1p-Ysl2p-Arl1p membrane remodeling complex
(2011) Barbosa, Sonia Cristina de Oliveira; Wolf, Dieter H. (Prof. Dr.)
Vesicular trafficking requires molecular mechanisms that drive membrane-enclosed organelles to bud vesicles and fuse with incoming ones. Understanding how these highly-curved transport structures are generated has been challenging. The best understood mechanisms for the generation of vesicles in the secretory and endocytic pathways involve the assembly of cytosolic coat proteins. However some other proteins have been implicated in the generation of the high curvature needed to form a vesicle. Prime candidates are the phospholipid translocases from the P4-ATPase subfamily, which are thought to pump phospholipids from the exocytosolic leaflet of a membrane to its cytosolic leaflet thereby deforming membranes. In the group of B. Singer-Krüger, the yeast P4-ATPase Neo1p was identified as being an interaction partner of Ysl2p, a protein that has sequence similarity to large Arf GEFs from the BIG and GBF family, and to be genetically linked to the small Arf-like GTPase Arl1p. A recent study demonstrated that a network formed by these proteins (the Neo1p-Ysl2p-Arl1p network), helps recruiting the GGAs clathrin adaptors to the TGN/endosomal system. Recently, members of the conserved Cdc50 family were identified as being transport chaperones for some of the P4-ATPases. Therefore, initially in this PhD thesis I studied the putative connection of Neo1p with this protein family. However, while all the other yeast P4-ATPases seem to require a specific member of the Cdc50 family for their export from the ER, this does not seem to be the case for Neo1p. This result led to the search for a novel protein required for Neo1p function. Herein, Dop1p, an essential yeast protein of about 195 kDa and highly conserved among eukaryotes, was identified as being a binding partner of Neo1p and Ysl2p. Studies using neo1, dop1, Δysl2 mutants, revealed that the respective proteins, Neo1p, Dop1p and Ysl2p, are interdependent thereby suggesting that they might exist in a complex. Significantly, in a temperature-sensitive dop1-3 mutant both Neo1p and Ysl2p were found to be specifically destabilised. Similarly, a comparable loss of Ysl2p and Dop1p was also found in neo1-69 cells while in Δysl2 cells a remarkable reduction of both Neo1p and Dop1p was observed. Consistent with this reciprocal dependency effect, NEO1 is a suppressor of dop1-3 and Δysl2 mutants and DOP1 a suppressor of neo1-69 and Δysl2 mutants. The relationship between Dop1p and Neo1p is further strengthened by the observation that the neo1-69 and dop1-3 mutants display several similar phenotypic defects and that similarly to Neo1p, Dop1p also localises to the endosomes. Interestingly, although Ysl2p interacts with Dop1p, the Neo1p-Dop1p interaction is independent of Ysl2p and Dop1p is still localised to the TGN/endosomes in the absence of Ysl2p. In order to get some insights in the features of the large protein Dop1, the conserved N-terminal region, the C-terminal region containing leucine zipper-like repeats, and the less conserved internal segment were separately expressed as GFP-fusions and their subcellular localisations and molecular interactions were analysed. These analyses revealed that the different regions of Dop1p had distinct localisation patterns and binding affinities to Neo1p and Ysl2p. The internal region of Dop1p seems to be particularly important for the endosomal membrane association of the protein. Additionally, the presence of leucine zipper-like repeats at the C-terminus of Dop1p suggested that this protein could form dimers or oligomers and indeed Dop1p is able to self-interact. In addition, a conserved PPSY motif in the C-terminus of Neo1p was identified. This led to the examination of Neo1p ubiquitination. The introduction of point mutations in this motif resulted in an improved cell growth at high temperatures. However, whether this motif is related to Neo1p ubiquitination awaits further investigations. In summary, this work identified Dop1p as a novel component of the Neo1p-Ysl2p-Arl1p complex. Moreover, the analysis of the distinct regions of Dop1p shed some light in their putative role within this protein.