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Functional analysis of syntaxin 16 phosphorylation using yeast as a model
Volfová, Barbora ; Entlicher, Gustav (advisor) ; Dráber, Petr (referee)
4 Abstract Mechanism of fusion of intracellular membranes in eukaryotic cells involves several protein families including soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins and Sec1/Munc-18 related proteins (SM proteins). It is known that the transport is evolutionary conserved from yeast to man. Therefore for facilitating of the research, we can use simple eukaryotes Saccharomyces cerevisiae. Mammalian SNARE protein syntaxin 16 has a yeast homologue Tlg2p which is used in this study as a model for studying affects of phosphorylation to the syntaxin 16 function. Also their binding partners, SM proteins mVps45p (mammalian) and yeast Vps45p are homologous. Phosphorylation of SNARE proteins is known as a possible way of regulation of membrane fusion. Abolishment of one of the putative phosphorylation sites in Tlg2p protein, serine 90 leads to dominant effects on the exocytic and endocytic pathways. The work presented in this study shows some phenotypes of mutants based on this phosphorylation site of protein Tlg2p. Those mutants are S90A (cannot be phosphorylated) and S90D (phosphomimetic - acid carboxyl group mimics phosphate group). It was revealed that the phosphorylation of Tlg2p protein at serine 90 or the mutation Tlg2p-S90D may play some role in protecting Tlg2p...
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Role of a SNARE protein in the biogenesis of Giardia intestinalis mitosomes.
Voleman, Luboš ; Doležal, Pavel (advisor) ; Nohýnková, Eva (referee)
SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...
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Sperm cell penetration and membrane fusion with oocyte during fertilization in mammals
Klimková, Veronika ; Krylov, Vladimír (advisor) ; Frolíková, Michaela (referee)
Fertilization is a process involving multiple steps, which are in continuity and complement each other. Spermatozoa become competent to fertilize after capacitation, hyperactivation and acrosome reaction. Oocytes are surrounded by a layer of cumulus cells and give a signals for spermatozoa to activation and orientation. This study focuses on the latest knowledges about a roles of cumulus cells, production of chemoattractants and the possibility that cumulus cells induce the acrosome reaction after binding a novel sperm protein NYD-SP8 to the cumulus. It seems that progesterone induce the acrosome reaction and also is the best chemoattractant secreted by cumulus cells. Next I am focus on gamete fusion including the role of surface proteins such as Juno and IZUMO1. Key words: membrane fusion, cumulus cells, acrosome reaction, chemotaxis sperm, oocyte, fertilization
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Membrane interactions studied by advanced fluorescent techniques: From ions to macromolecules
Pokorná, Šárka ; Hudeček, Jiří (advisor) ; Konopásek, Ivo (referee) ; Benda, Aleš (referee)
Advanced fluorescence techniques were used to explore tree distinct topics concerning biological membrane and their interactions. Following thesis is according to the topic divided into three parts: 1) Ionic effects were studied employing time dependent fluorescence shift experiments and molecular dynamic simulations. Combination of these two approaches are suitable to reveal characteristic like mobility and hydration of particular bilayer segment, lipid packing or ion binding sites. Halide anions were reported to adsorb to the cationic lipid bilayer specifically, altering membrane mobility and organization. Changes in observed parameters follows Hofmeister order. Their effect is mediated either by direct ionic interaction (soft, polarizable ions) as well as via alteration of water structure (hard, non-polarizable ions) in proximity of ion molecule. Further, divalent calcium was shown to bind strongly to neutral and negatively charged lipid bilayers. Several types of binding sites depending on calcium concentration were identified. 2) Two complementary lipopeptides, CPK and CPE, incorporated into distinct lipid bilayers serve as a minimal model inducing membrane fusion. Effectiveness of fusion event might be influenced by lipopeptide-membrane and lipopeptide-lipopeptide interaction. To reveal...
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Role of a SNARE protein in the biogenesis of Giardia intestinalis mitosomes.
Voleman, Luboš ; Doležal, Pavel (advisor) ; Nohýnková, Eva (referee)
SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...
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Functional analysis of syntaxin 16 phosphorylation using yeast as a model
Volfová, Barbora ; Entlicher, Gustav (advisor) ; Dráber, Petr (referee)
4 Abstract Mechanism of fusion of intracellular membranes in eukaryotic cells involves several protein families including soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins and Sec1/Munc-18 related proteins (SM proteins). It is known that the transport is evolutionary conserved from yeast to man. Therefore for facilitating of the research, we can use simple eukaryotes Saccharomyces cerevisiae. Mammalian SNARE protein syntaxin 16 has a yeast homologue Tlg2p which is used in this study as a model for studying affects of phosphorylation to the syntaxin 16 function. Also their binding partners, SM proteins mVps45p (mammalian) and yeast Vps45p are homologous. Phosphorylation of SNARE proteins is known as a possible way of regulation of membrane fusion. Abolishment of one of the putative phosphorylation sites in Tlg2p protein, serine 90 leads to dominant effects on the exocytic and endocytic pathways. The work presented in this study shows some phenotypes of mutants based on this phosphorylation site of protein Tlg2p. Those mutants are S90A (cannot be phosphorylated) and S90D (phosphomimetic - acid carboxyl group mimics phosphate group). It was revealed that the phosphorylation of Tlg2p protein at serine 90 or the mutation Tlg2p-S90D may play some role in protecting Tlg2p...
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