Abstract
The asymmetric distribution of phospholipids in biological membranes is tightly controlled by several families of membrane-embedded proteins. One of these includes the P4-ATPases, which are able to perform the transport or “flip” of phospholipids from the exoplasmic- to the cytoplasmic leaflet, hence their nickname, “flippases”. These transporters are exclusively found in eukaryotic organisms, where they are involved in lipid-based signaling events such as blood, coagulation, apoptosis and vesicular biogenesis. The P-type ATPases are an essential superfamily of primary transporters that ‘pump’ several different cations and lipids across cellular membranes, a process driven by adenosine triphosphate (ATP). These pumps have been extensively studied both biochemically and structurally throughout the last decades. However, flippases remain among the least understood members, especially with regard to its much larger substrate and how the mechanism allowing the transport unfolds. This is one of the central questions in the field known as the “giant substrate problem”. To this date, no structural information of P4-ATPases is available.
The focus of this thesis is divided into two projects, both focusing on P4-ATPases from the yeast organism Saccharomyces cerevisiae:
I. The structural characterization of the flippase Drs2p in complex with its auxiliary subunit Cdc50p.
II. Establishing a protocol for obtaining a homogenous sample of the flippase Neo1p suitable for biochemical characterization and substrate identification.
Part I was performed using X-ray crystallography and single-particle electron microscopy as the main methods. A 3D envelope was obtained by cryo-EM extending to a resolution of 4.4 Å. This envelope reveals the first structural insight of the conformational organization of the Drs2p+Cdc50p complex.
Part II yielded a growth- and purification protocol for obtaining a homogenous sample of recombinantly expressed Neo1p in detergent. However, initial attempts to stimulate ATP-turnover in the presence of putative substrates were unsuccessful. Work is currently on going to elucidate potential regulatory mechanisms of Neo1p, in order to achieve substrate specific activity.
The focus of this thesis is divided into two projects, both focusing on P4-ATPases from the yeast organism Saccharomyces cerevisiae:
I. The structural characterization of the flippase Drs2p in complex with its auxiliary subunit Cdc50p.
II. Establishing a protocol for obtaining a homogenous sample of the flippase Neo1p suitable for biochemical characterization and substrate identification.
Part I was performed using X-ray crystallography and single-particle electron microscopy as the main methods. A 3D envelope was obtained by cryo-EM extending to a resolution of 4.4 Å. This envelope reveals the first structural insight of the conformational organization of the Drs2p+Cdc50p complex.
Part II yielded a growth- and purification protocol for obtaining a homogenous sample of recombinantly expressed Neo1p in detergent. However, initial attempts to stimulate ATP-turnover in the presence of putative substrates were unsuccessful. Work is currently on going to elucidate potential regulatory mechanisms of Neo1p, in order to achieve substrate specific activity.
| Translated title of the contribution | Strukturel og funktionel karakterisering af P4-ATPase lipid flippaser |
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| Original language | English |
| Number of pages | 129 |
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| Publication status | Published - 23 Mar 2018 |