Godina izdanja: Ostalo
ISBN: Ostalo
Oblast: Biologija
Jezik: Engleski
Autor: Strani

56422-SN) Structural studies of the yeast lipid flippase , Drs2p / Cdc50p , using cryo – electron microscopy PhD Thesis ,
Author : Milena Timcenko Department of Molecular Biology and Genetics Aarhus University
Abstract (English)

Phospholipids are a major building block of cellular membranes. A hallmark of the late secretory pathway of eukaryotes is the presence of an asymmetric distribution of lipids between the two leaflets. The regulation of the lipid distribution is important for many biological processes, such as cell-to-cell signaling, vesicle formation and apoptosis.

One of the least understood and characterized members of the P-type ATPase family, are the P4 ATPases or `lipid flippases`. These transporters are only present in eukaryotes, and utilize the energy from ATP hydrolysis to fuel unidirectional flipping of phospholipids across the membrane bilayer, from the extra- to the intracellular leaflet. Hereby they contribute to establishing and maintaining the asymmetric lipid distribution.

A central question in the study of P4 ATPases is, how they are able to accommodate and transport a much larger substrate than other P-type ATPases, which usually transport small cations. This is known as the `giant substrate problem of the lipid flippases.

With the aim of studying the structure, transport mechanism and dynamies of lipid flip pases, the work presented in this thesis focuses on the phosphatidylserine (PS) specific het erodimeric flippase Drs2p/Cdc50p from the Trans-Golgi network (TNG) of Saccharumpoes cerevisiac. Drs2p, the P4 ATPase, has an auto-inhibitory domain in its C-terminus, where binding of non-substrate lipid phosphatidylinositol-4-phosphate (P14P) is able to release this inhibition in vitro.

Drs2p/Cdc50p was over-expressed in S. cerevision and purified in detergents DDM and LMNG, as well as reconstituted into amphipol A8-35 and salipro nanoparticles. This was done with the goal of determining the structure of Drs2p/Cde 50p by cryo-electron mi crocopy. The PS-specific ATPase activity was retained in all four environments, under conditions where the auto-inhibition is released by PHP Phosphate and nucleotide ana gue inhibitors were studied for the purpose of locking specific conformational states of Dodp/Cdc50p for structural studies.

Single particle cryo-EM studies of Drs2p/Cdc30p in LMNG resulted in three structure at of2A, 17A and 20A. They map progressive stages of activation, from alt auto ulubited structure, ver an intermediate PHP-bound state to a hilly activated PEPbound state of a Ceritally trunrated enzyme Preliminary cryo FM investigations into other mation of Divip/Ciclip have further been initiated

Thesh agent ulatrate is onered, the work in this thesis rejeede the very fast P ATP d Ripp well as the first structure of an auto-inhibited dation of Pippe ATPas
Papaerback, size 17,5 x25 cm , english language , 230 pages, signed by author on title page , Illustrated