E-Locus - Institutional Repository of the University of Crete

Home Collections Type of Work Post-graduate theses

Post-graduate theses

Search command : Author="Στεφανίδης" And Author="Κωνσταντίνος"

Current Record: 4597 of 6513

[Add to Basket]

Identifier

000376319

Title

Γενετική και βιοχημική ανάλυση της οξειδωτικής αναδίπλωσης πρωτεϊνών στα μιτοχόνδρια

Alternative Title

Genetic and biochemical analysis of the oxidotine folding of proteins in mitochondria

Author

Κάλεφ- Εζρά, Έστερ Τζω.

Thesis advisor

Τοκατλίδης, Κώστας
Αλεξανδράκη, Δέσποινα

Abstract

Most mitochondrial proteins are encoded by nuclear genes and translated in the cytoplasm before entering in mitochondria. Some proteins of the intermembrane space (IMS) of mitochondria use the intermembrane space import and assembly machinery (MIA) pathway to be inserted into mitochondria. The protein substrates for this pathway are oxidized by the protein Mia40 after crossing the outer membrane and are trapped into the IMS. Subsequently, the sulfhydryl oxidase Erv1/ALR reoxidizes Mia40 with electrons finally transferred to either O₂ or cytochrome-c. In this study: 1. Quantitative changes in the proteins Mia40, Erv1 and cyt-c and cell viability were studied in yeast S. cerevisiae and isolated mitochondria upon oxidative stress. Higher sensitivity to oxidative stress was observed in cells and their mitochondrial proteins grown in a medium metabolized in the cytoplasm than that in those grown in a medium metabolized in mitochondria. 2. We attempted to identify in Mia40 and Erv1/ALR the existence of evolutionary conserved motif near the catalytic disulfides. Short-forms and long-forms due to alternative splicing were found only in the ALR proteins of mammals. 3. The R-rich motif (Rx4Rx4Rx6Rx4R) of human ALR, a conserved motif of unknown function, was mutated to alanines for future studies on the importance of the compartmentalization of the two forms of the ALR in human cells. 4. The ability of the first 72 amino acids of the protein of the yeast protein Erv1 (N72) regarding to introduce non mitochondrial proteins (DHFR, NHP6A) in mitochondria was studied. Little or no input signal was found under the employed experimental conditions. 5. Yeast Erv1 and human ALR were mutated to study in detail the intracellular targeting process of these proteins. Specifically, we mutated yeast Erv1 isoleucine at position 22 (I22) to lysine and aspartic acid. It was found under such conditions, the import of non mitochondrial DHFR in the mitochondria via the first 72 amino acids of Erv1 is facilitated. Also we mutated the R-rich motif of the human ALR to alanines to investigate the importance of this motif in future studies. Similarly, the plasmid constructs pEGFP-N1-N72 (Erv1), pEGFP-N1-cyt-b2 (N85), pEGFP-N1-Erv1, pEGFP-N1-N80 (ALR), pEGFP-N1-sf-ALR were created to be used in future studies on the ability of the proteins to compartmentalize ALR human (lf-ALR, sf-ALR) and S. cerevisiae Erv1 in human cell lines.

Language

Greek

Subject

Import

Pathway

Είσοδος

ΜΙΑ

Μονοπάτι

Issue date

2012-11-16

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses