TY - BOOK

T1 - Purification and biochemical characterisation of the yeast ABC transporter Pdr11p

T2 - towards unravelling the sterol transport mechanism

AU - Laub, Katrine Rude

PY - 2017

Y1 - 2017

N2 - Sterols constitute an essential lipid class in eukaryotic membranes where intracellular distributions are highly regulated. In the yeast Saccharomyces cerevisiae sterol uptake has been attributed to the two plasma membrane-localised ATP-binding cassette (ABC) transporters, Aus1p and Pdr11p. These ATP-powered transporters are proposed to mediate inter- and transbilayer sterol migration, but their exact role and key features of their activity remain to be elucidated. The complexity of the native membrane hampers the study of membrane transporters at the molecular level. To achieve a detailed understanding of their action and molecular functioning, it is helpful to focus on homogeneous preparations of membrane proteins reconstituted into model membranes like detergent micelles, liposomes, and nanodiscs. In the present study, I report the first successful expression, purification, and reconstitution of the yeast ABC transporter Pdr11p. This includes optimising its overexpression utilising the galactose induction system in S. cerevisiae, screening for the best detergent to extract the protein from the membrane, and establishing purification and reconstitution protocols. By providing a purification/reconstitution protocol and adding new insight to the biochemical behaviour of Pdr11p this work paves for future studies to unravel the sterol transport mechanism.

AB - Sterols constitute an essential lipid class in eukaryotic membranes where intracellular distributions are highly regulated. In the yeast Saccharomyces cerevisiae sterol uptake has been attributed to the two plasma membrane-localised ATP-binding cassette (ABC) transporters, Aus1p and Pdr11p. These ATP-powered transporters are proposed to mediate inter- and transbilayer sterol migration, but their exact role and key features of their activity remain to be elucidated. The complexity of the native membrane hampers the study of membrane transporters at the molecular level. To achieve a detailed understanding of their action and molecular functioning, it is helpful to focus on homogeneous preparations of membrane proteins reconstituted into model membranes like detergent micelles, liposomes, and nanodiscs. In the present study, I report the first successful expression, purification, and reconstitution of the yeast ABC transporter Pdr11p. This includes optimising its overexpression utilising the galactose induction system in S. cerevisiae, screening for the best detergent to extract the protein from the membrane, and establishing purification and reconstitution protocols. By providing a purification/reconstitution protocol and adding new insight to the biochemical behaviour of Pdr11p this work paves for future studies to unravel the sterol transport mechanism.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122333691105763

M3 - Ph.D. thesis

BT - Purification and biochemical characterisation of the yeast ABC transporter Pdr11p

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -