Cdc50p Plays a Vital Role in the ATPase Reaction Cycle of the Putative Aminophospholipid Transporter Drs2p

Guillaume Lenoir,Patrick Williamson,Catheleyne F Puts,Joost C.M Holthuis

doi:10.1074/jbc.m109.013722

Guillaume Lenoir, Patrick Williamson + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.013722

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Abstract

Members of the P(4) subfamily of P-type ATPases are believed to catalyze transport of phospholipids across cellular bilayers. However, most P-type ATPases pump small cations or metal ions, and atomic structures revealed a transport mechanism that is conserved throughout the family. Hence, a challenging problem is to understand how this mechanism is adapted in P(4)-ATPases to flip phospholipids. P(4)-ATPases form heteromeric complexes with Cdc50 proteins. The primary role of these additional polypeptides is unknown. Here, we show that the affinity of yeast P(4)-ATPase Drs2p for its Cdc50-binding partner fluctuates during the transport cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. We also find that specific interactions with Cdc50p are required to render the ATPase competent for phosphorylation at the catalytically important aspartate residue. Our data indicate that Cdc50 proteins are integral components of the P(4)-ATPase transport machinery. Thus, acquisition of these subunits may have been a crucial step in the evolution of flippases from a family of cation pumps.

Highlights

Cyclic changes between two main enzyme conformations, E1 and E2, during which the ATPase is phosphorylated by ATP at the aspartate residue and subsequently dephosphorylated
H2-Drs2p expressed in wild type cells primarily localized to the endoplasmic reticulum (ER), yet co-expression of CDC50-Myc allowed a substantial portion of H2-Drs2p to reach the Golgi complex (Fig. 1D)
Dnf1p had the same effect as observed for Drs2p: a substantial reduction in interaction between transporter and subunit. These findings suggest that a phosphorylatable active site is a common prerequisite for stability of P4-ATPaseCdc50 complexes in vivo and that the interaction between transporter and subunit may fluctuate during the reaction cycle

Summary

EXPERIMENTAL PROCEDURES

Plasmids, and Yeast Strains—All yeast strains, plasmids, antibodies, and reagents used in this study are described in the supplemental data. After centrifugation at 18,000 gav (4 °C, 25 min), protein pellets were washed with 7% trichloroacetic acid and 0.5 mM H3PO4, resuspended in 40 ␮l of SDS sample buffer (2% SDS, 10 mM EDTA, 150 mM Tris-HCl, pH 6.8, 16% (v/v) glycerol, 0.8 M ␤-mercaptoethanol, 0.04% bromphenol blue), and loaded onto acidic Sarkadi-type gels for electrophoretic separation. Constructs containing C terminus of ubiquitin (Cub) were made by in vivo recombination in the yeast strain THY.AP4 (MATa ura leu lexA::lacZ::trp lexA::HIS3 lexA::ADE2). Constructs containing the N terminus of ubiquitin (Nub) were made by in vivo recombination in the yeast strain THY.AP5 (MAT␣ URA3 leu trp his loxP::ade). The diploids were replicated on SD-Leu-Trp-His-Ade plates to test for growth Sensitivity of these growth assays was determined by Met-controlled expression of the Cub construct. After incubation at 30 °C for 10 min, the assay mixture was sedimented, and the A420 of the supernatant was measured to obtain specific activity for ␤-galactosidase

RESULTS

Coupled to the ATPase Reaction

If phosphorylation is required for robust interaction with

DISCUSSION