Browsing by Author "Lasic, Maja"

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    The yeast endosomal/TGN-localized Ysl2p-Arl1p-Neo1p network: search for novel interaction partners
    (2008) Lasic, Maja; Singer-Krüger, Birgit (PD Dr.)
    Vesicle transport is crucial for the communication between the compartments of a eukaryotic cell during the internalisation of endocytosed material, secretion of proteins into the extracellular space and biosynthetic transport to the vacuole. During each transport step the budding, tethering and fusion of the vesicle has to be regulated by numerous proteins to ensure the specificity of transport. Arfs and Rabs, small GTPases of the Ras superfamily, are crucial switches in these processes due to their GDP/GTP cycle. Arf GTPases regulate mainly the budding of vesicles by recruiting adaptor proteins to membranes, which in turn ensure the packaging of appropriate cargo into the vesicle and enable the coat assembly. Ysl2p is a large protein, which has in the group of Dr. Birgit Singer-Krüger been shown to play a role in both the endocytic and biosynthetic route. Sequence analysis has further revealed homologies of Ysl2p to Sec7 family Arf GEFs. Due to this homology and the interaction of the N-terminus of Ysl2p, which contains the Sec7 domain, with the Arf-like GTPase Arl1p, Ysl2p was proposed to be a guanine nucleotide exchange factor for Arl1p. ARL1 was as well identified as a suppressor of the growth defect of Δysl2 cells. Arl1p localises to TGN and endosomes, where it recruits GRIP domain proteins to the membrane. Since GRIP-domain proteins are regarded as regulators of vesicle tethering, Arl1p was also proposed to be mainly involved in the tethering of vesicles. The NEO1 gene, which encodes an aminophospholipid translocase, has been identified as a second suppressor of the Δysl2 mutant. Same study of the Dr. B. Singer-Krüger group has demonstrated an in vivo interaction between Neo1p and Ysl2p. Thus, Neo1p is together with Ysl2p and Arl1p a further component of a common network. The work presented in this PhD thesis focussed on the role of Ysl2p within this network. Former analysis from the Dr. B. Singer-Krüger group have demonstrated strong diferencies between some protein levels of Δysl2- and wild type cells when compared by one-dimensional electrophoresis. In the present study, the protein composition of vacuoles isolated from Δysl2- and wild type cells were compared using two dimensional electrophoresis. Subsequently, the differing proteins were analysed by mass spectroscopy (collaboration with Dr. A. Sickmann group, Würzburg) to identify which protein levels are affected by the deletion. From all observable changes upon YSL2 deletion, the most prominent was the increase of the vacuolar levels of Erg6p. Additionally, a reduction of the vacuolar levels of some of the V-ATPase subunits and actin could be observed in Δysl2 cells. Subcellular fractionation experiments were performed to analyse if the changes of vacuolar levels of the V-ATPase were accompanied by changes in its distribution between TGN and endosomes. Additionally, other marker proteins of the TGN and endosomes were analysed for their distribution. However, no significant changes in the distribution of the analysed proteins could be observed. Interestingly, the cellular levels of Kex2p were significantly reduced in Δysl2 cells, which indicates on a possible role for Ysl2p in sorting of Kex2p on TGN/endosomes. For Ysl2p the subcellular distribution indicates a primarily early endosomal localisation. To identify novel interaction partners of Ysl2p, a two hybrid screen was performed in collaboration with the group of Dr. P. Uetz (Karlsruhe) with different subdomains of this large protein. Surprisingly, although several putative interaction partners of Ysl2p were identified in the screens, only the putative interactors of the central region of the protein (Ysl2PILT) could be confirmed in my subsequent studies. Ent4p appears to be the most interesting candidate since its homologues Ent3p and Ent5p have recently been found to interact with Gga2p, a putative binding partner of Ysl2p. A weak interaction of Ent4p and Ysl2p could be detected by a GST-pull down but not by a co-immunoprecipitation experiment. An alternative approach to identify novel interaction partners of Ysl2p was a large scale immunoprecipitation of Ysl2p-TAP. Although Ysl2p could be enriched with high purity no additional band could be specifically co-purifed by Ysl2p-TAP. This is surprising, since in a similar study Gillingham et al. (2006) isolated the protein Dop1p equimolar to Ysl2p, suggesting that the two proteins exist in a complex. Possible explanation for the failure in the present study could be a false purification method, since cells were lysed by grinding under liquid nitrogen while Gillingham et al. used spheroblasts for the detection. To analyse the importance of different domains of the Ysl2p protein, a deletion series was generated, in which 100 to 900 amino acids from the C-terminus of Ysl2p were exchanged by the TAP-epitope and the corresponding effect on the cell growth was analysed. Interestingly, already the deletion of C-terminal 100 amino acids caused the same growth defect as the deletion of the complete YSL2 gene. Further, it was demonstrated by a co-immunoprecipitation experiment that Ysl2p interacts with itself and that the C-terminal 100 amino acids are crucial for this interaction. Thus, the dimerisation of Ysl2p by the C-terminus could be highly important for cellular function. Former study from the group of Dr. B. Singer-Krüger analysed the role of the APL translocase Neo1p within the Ysl2p-Arl1p network. There it was demonstrated, that at restrictive temperatures the temperature-sensitive neo1-69 mutant accumulates aberrant membrane protrusions within the cell. Interestingly, the same study showed that the deletion of the ARL1 gene restores the growth of the neo1-69 mutant at the restrictive temperature, possibly by preventing the accumulation of Arl1p and effectors at the aberrant membrane protrusions. To identify possible Arl1-effectors, the experiment was repeated in the present study with deletions of several known TGN/endosomal adaptors. While the deletion of GGA1 had no effect on the growth of the neo1-69 mutant and the deletion of AP-1 and AP-3 subunits caused only partial suppression, the deletion of the adaptor GGA2 could suppress the neo1-69 growth defect to the same extent as the deletion of ARL1. Analogous analysis with several small GTPases demonstrated that, while the deletion of Ypt GTPases YPT7 and YPT51 had no effect on the growth of the neo1-69 mutant, the deletion of ARF1 caused partial and deletion of ARL3 caused suppression comparable to the deletion of ARL1. The connection between Gga2p and the Ysl2-Arl1-Neo1 network indicated by genetical analysis was supported by indirect immunofluorescence analysis, which demonstrated that the deletions of either ARL1 or YSL2 caused a loss of punctuate structures in the Gga2p-HA staining while in some neo1-69 mutant cells an accumulation of Gga2p in the aberrant structures could be observed. Further, a genetic interaction of ARL1 with the GGAs as well as an interaction of the VHS-GAT domain of Gga2p with Arl1p and Ysl2p by a GST-pull down experiment could be demonstrated. Thus, Arl1p and its network seem to play a role not only in tethering as indicated by former analysis but as well in vesicle budding together with the adaptor protein Gga2p. Finally, the protein Tvp38p has earlier been demonstrated to interact with Ysl2p in a two hybrid assay and a co-immunoprecipitation. Interestingly, Miller et al., (2005) have recently demonstrated an interaction between Sys1p and Tvp38p in a split ubiquitin screen. Both interactions could be confirmed in the present study, the Ysl2p-Tvp38p interaction by a GST-pull down assay and the Sys1p-Tvp38p interaction by a co-immunoprecipitation experiment. Thus, Tvp38p appears to be a link between the networks Ysl2-Arl1-Neo1 and Sys1-Arl3-Arl1. That could be a hint for a common role of these proteins. In summary, the present study provides evidence that the Ysl2-Arl1-Neo1 network plays an important role in membrane recruitment of the adaptor Gga2p to membranes and thus may participate in vesicle budding processes. Further, Ysl2p may play a role in the localisation of Erg6p, which regulates one of the final maturation steps in ergosterol synthesis.