Ins and Outs of a Heterodimeric Phospholipid Pump

R. Panatala Narendranath

Research output: ThesisDoctoral thesis 1 (Research UU / Graduation UU)


Type 4 P-type ATPases (P4-ATPases) catalyze phospholipid transport to create lipid asymmetry across membranes of late secretory and endocytic compartments. P4-ATPases evolved from an ancient family of cation pumps, the P-type ATPase superfamily, which includes Ca2+-ATPases, Na+/K+-ATPases, H+-ATPases and various heavy-metal transporters. Crystal structures and detailed functional analyses of different cation-transporting P-type ATPases revealed a transport mechanism that appears to be conserved throughout the family. A key challenge is to understand how this mechanism is adapted by P4-ATPases to flip bulky phospholipids. Notably, phospholipids are ~10-fold larger than the ligands of cation-transporting P-type ATPases and have to reorient during their translocation. This enigma has been referred to as the “giant substrate problem”. In this thesis, we used two complementary approaches to elucidate the role of Cdc50 subunits in P4-ATPase-catalyzed phospholipid transport and gain further insight into the inner workings of heterodimeric flippases. As first approach, we set out to identify structural determinants that govern functional interactions between subunit and transporter in yeast. To this end, we took advantage of separate assays for binding and activity, allowing a molecular dissection of the relationship between Cdc50 binding and P4-ATPase-catalyzed phospholipid transport. As a complementary approach, we established a liposome-coupled, cell-free expression system to allow the production and functional analysis of P4-ATPases independently of their Cdc50 binding partner.
Original languageEnglish
Awarding Institution
  • Utrecht University
  • Holthuis, J.C.M., Primary supervisor
  • van Meer, Gerrit, Supervisor
Award date9 Dec 2014
Place of PublicationUtrecht
Print ISBNs978-90-5335-977-8
Publication statusPublished - 9 Dec 2014


  • Lipid asymmetry
  • P4-ATPases
  • Cdc50 proteins
  • giant substrate problem
  • cell-free translation


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