Abstract
Members of the P(4) subfamily of P-type ATPases catalyze phospholipid transport and create membrane lipid asymmetry in late secretory and endocytic compartments. P-type ATPases usually pump small cations and the transport mechanism involved appears conserved throughout the family. How this mechanism is adapted to flip phospholipids remains to be established. P(4)-ATPases form heteromeric complexes with CDC50 proteins. Dissociation of the yeast P(4)-ATPase Drs2p from its binding partner Cdc50p disrupts catalytic activity (Lenoir, G., Williamson, P., Puts, C. F., and Holthuis, J. C. (2009) J. Biol. Chem. 284, 17956-17967), suggesting that CDC50 subunits play an intimate role in the mechanism of transport by P(4)-ATPases. The human genome encodes 14 P(4)-ATPases while only three human CDC50 homologues have been identified. This implies that each human CDC50 protein interacts with multiple P(4)-ATPases or, alternatively, that some human P(4)-ATPases function without a CDC50 binding partner. Here we show that human CDC50 proteins each bind multiple class-1 P(4)-ATPases, and that in all cases examined, association with a CDC50 subunit is required for P(4)-ATPase export from the ER. Moreover, we find that phosphorylation of the catalytically important Asp residue in human P(4)-ATPases ATP8B1 and ATP8B2 is critically dependent on their CDC50 subunit. These results indicate that CDC50 proteins are integral part of the P(4)-ATPase flippase machinery.
Highlights
Project). 3 To whom correspondence should be addressed: Membrane Enzymology, 6), while its regulated dissipation is associated with a multitude ofphysiological processes, including blood coagulation, sperm capacitation, myoblast fusion, virus entry into host cells, and phagocytic clearance of apoptotic bodies [7,8,9]
For P4ATPases we focused on the six members of the ATP8 or class-1 cluster, i.e. ATP8A1, ATP8A2, ATP8B1, ATP8B2, ATP8B3, and ATP8B4
When expressed at very high levels, these P4-ATPases showed a nuclear-envelope and reticular staining that coincided with the endoplasmic reticulum (ER) marker protein disulfide isomerase (PDI; Fig. 2A, ATP8B4-HA-positive cell marked with an asterisk)
Summary
5Ј-aaaagcagtcctcttgataacct-3Ј 5Ј-ttgctatcccgcagcaccgcttt-3Ј 5Ј-tcagctgtccccggtgc-3Ј 5Ј-gccaacccgccgcatgcagcg-3Ј 5Ј-tgggctcgaagctgcctc-3Ј 5Ј-gctaaagctgctcacggtg-3Ј 5Ј-aatggtaatgtcagctgtattac-3Ј 5Ј-ataaactggtaagatgatatcac-3Ј 5Ј-gcactttgatttgtacagatga-3Ј. Human ATP8B1, a P4-ATPase linked to familial intrahepatic cholestasis or Byler disease [37], requires a CDC50 homologue for ER export and delivery to the plasma membrane [38] Whereas these studies clearly demonstrate that CDC50 proteins are indispensable for proper intracellular targeting of P4-ATPases, they do not address whether CDC50 proteins contribute to the transport properties of the complex. Using a genetic reporter system, we found that the affinity of Drs2p for Cdc50p fluctuates during the reaction cycle with the strongest interaction occurring at or near a point where the enzyme is loaded with phospholipid ligand [39] Together, these results suggest that CDC50 proteins play a critical role in the P4-ATPase transport reaction. To gain further insight into the role of CDC50 proteins in P4-ATPase-catalyzed phospholipid transport, we here set out to systematically map physical and functional interactions between human class-1 P4-ATPase and CDC50 family members
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