Vloga gena PEX11 in identifikacija novih genov z vplivom na lipidni metabolizem kvasovke Saccharomyces cerevisiae
| Autor: | Ogrizović, Mojca |
|---|---|
| Přispěvatelé: | Petrovič, Uroš |
| Jazyk: | slovinština |
| Rok vydání: | 2019 |
| Předmět: |
proteinske interakcije
organelle interaction genetic pathways medorganelno stiki poligenska lastnost protein interactions polygenic trait kvantitativni lokus metabolizem lipidov quantitative trait locus udc:602.3:582.282.23:601.4:577.21(043.3) lipid metabolism pleiotropy peroxisomal membrane protein pleitrofnost membranski protein peroksisomov genetske poti Pex11 |
| Popis: | Peroksisomi in lipidne kapljice so ključni organeli pri vzdrževanju homeostaze metabolizma lipidov v kvasovki Saccharomyces cerevisiae. Na strani peroksisomov smo preučevali membranski protein Pex11, ki je potreben za elongacijo in cepitev peroksisomov, vpliva pa tudi na izražanje genov glikolize. Na strani lipidnih kapljic smo ovrednotili doprinos kandidatnih vzročnih genov MKT1, PHO23, PIG1 in RML2 na kopičenje založnih lipidov in iskali morebitne dodatne genetske elemente tega fenotipa. Z membranskim dvo-hibridnim sistemom kvasovke in bi-molekularnim fluorescenčnim dopolnjevanjem smo odkrili interakcijo med proteinom Pex11 in membranskim proteinom mitohondrijev, Mdm34. Z uporabo fluorescenčnih označevalcev za peroksisome in mitohondrije smo ugotovili, da je interakcija Pex11-Mdm34 pomembna za oblikovanje sidra PerMES (Peroksisom-ERMES), potrebnega za neposredno bližino peroksisomov in mitohondrijev. Da bi pojasnili povezavo osrednjega in lipidnega metabolizma preko gena PEX11, smo merili jakost izražanja genov za encime glikolize in ugotovili, da gen RLF2 ojača izražanje gena za enolazo ENO1, pri čemer je gen PEX11 ključen. Z zamenjavami alelov kandidatnih vzročnih genov med sevoma z različno vsebnostjo založnih lipidov ter z inaktivacijo teh genov v obeh sevih smo pokazali, da so aleli PHO23AWRI, PIG1AWRI in RML2S288c superiorni za stopnjo kopičenja založnih lipidov. S povratnim križanjem z obema sevoma pa smo odkrili dodatne genetske elemente, ki potencialno pripomorejo h kopičenju založnih lipidov. Raziskava je pomembno prispevala k razumevanju vloge proteina Pex11, ki je neodvisna od proliferacije peroksisomov, ter osvetlila genetsko arhitekturo kopičenja založnih lipidov v kvasovki S. cerevisiae. Peroxisomes and lipid droplets are key organelles in lipid metabolism in yeast Saccharomyces cerevisiae. We studied the role of a peroxisomal membrane protein Pex11, which is involved in the proliferation of peroxisomes and affects the expression of glycolytic genes. Regarding lipid droplets, the involvement of candidate causative genes MKT1, PHO23, PIG1 and RML2 in neutral lipid accumulation was evaluated and additional genetic elements affecting neutral lipids were analyzed. Using membrane yeast two hybrid system and bimolecular fluorescence complementation, we revealed an interaction between the proteins Pex11 and a mitochondrial membrane protein, Mdm34. Additionally, using fluorescent markers for peroxisomes and mitochondria, we proved that this interaction established a protein tether PerMES (Peroxisome-ERMES) between mitochondria and peroxisomes. By measuring the expression level of genes encoding glycolytic enzymes, we demonstrate association between central carbon metabolism and lipid metabolism through PEX11 gene: the gene RLF2 enhances the expression of ENO1, encoding enolase, and this effect is dependent on the presence of functional PEX11. By performing allele swaps of candidate causative alleles between two strains accumulating different levels of neutral lipids, and by constructing deletion mutants of these genes in both strains, we proved the alleles PHO23AWRI, PIG1AWRI in RML2S288c as superior for neutral lipid accumulation. We revealed potential additional genetic elements of neutral lipid accumulation by backcrossing to both parental strains. Importantly, our research contributes to the understanding of the role of Pex11 in processes other than peroxisomal proliferation, and elucidates the genetic architecture of neutral lipid accumulation in S. cerevisiae. |
| Databáze: | OpenAIRE |
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