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Interaction of protein subunits SEC10 and SEC15 of the exocyst tethering complex
Bartáková, Anna ; Ryšlavá, Helena (advisor) ; Čermáková, Michaela (referee)
The exocyst is an evolutionarily conserved tethering complex involved in the regulation of the secretory pathway in eukaryotic cells. As an effector of Small GTPases, the exocyst contributes to efficient targeting of secretory vesicles to the sites of intense exocytosis at the plasmatic membrane through interactions with specific membrane phospholipids. The exocyst complex consists of eight subunits: SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84. Each subunit binds at least two other subunits, among them the interaction of SEC10 and SEC15 is crucial for the exocyst function. The question remains how this particular interaction is evolutionarily conserved across different plant taxonomic groups. SEC10 and SEC15 genes from the three evolutionary groups of plants (green algae - Klebsormidium nitens, mosses - Marchantia polymorpha, angiosperms - Arabidopsis thaliana) were cloned into yeast two-hybrid vectors for studying protein interactions. Testing the interaction of SEC10 and SEC15 exocyst subunits from different plant species in the yeast-two hybrid system showed that despite partially different protein sequences of the tested subunits, their interaction across different evolutionary groups of plants remains very well conserved.
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Searching for interaction partners associated with the transcriptional apparatus of yeast linear plasmids
Ľalíková, Kristýna ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
The pGKL plasmids are a type of yeast linear double-stranded DNA plasmids found in the cytoplasm of yeast Kluyveromyces lactis. The plasmid gene transcription process involves the plasmid transcription apparatus, which shows similarity to the transcription apparatus of poxviruses. Poxviruses cause a range of serious diseases. Thus pGKL plasmids offer a safe way to study some of the mechanisms of these viruses without the risk of infection. The plasmid transcription apparatus comprises non-canonical RNA polymerase, helicase, and capping enzyme as its main components. Although RNA polymerase has been thoroughly characterized and described, little focus has been on the interactions between proteins within the transcription apparatus. Therefore, this study aims to shed light on this fundamental aspect of the gene expression of pGKL plasmids. The main goal of the thesis was to prepare a system that would facilitate the search for interaction partners of the pGKL transcription apparatus and verify the interactions between chosen proteins in yeast cells. The foundation of this system relies on the use of optimized genes and the yeast two-hybrid system method. The development of the experimental method has begun successfully, and it's expected to conclude soon, although further optimizations are still...
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