Heteromeric Interactions Required for Abundance and Subcellular Localization of Human CDC50 Proteins and Class 1 P4-ATPases

Abstract

Members of the P 4 family of P-type ATPases (P 4 -ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P 4 -ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P 4 -ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P 4 -ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P 4 -ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P 4 -ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.

ATP9B, a P4-ATPase (a Putative Aminophospholipid Translocase), Localizes to the trans-Golgi Network in a CDC50 Protein-independent Manner

“…2, I and J, and supplemental Fig. S1) as reported previously (24,25). By contrast, the staining for class 2 P4-ATPases (ATP9A and ATP9B) was observed on intracellular organelles (Fig.…”

“…In contrast, only a few P4-ATPases were translocated upon coexpression of CDC50B: ATP8B1 was localized to the plasma membrane as seen with CDC50A coexpression (Fig. 2JЉ) (23)(24)(25) and ATP11C was localized to the plasma membrane but to a much lesser extent (Fig. 2, HЈ and HЉ).…”

“…Translocation of P4-ATPases from ER upon Exogenous Expression of CDC50A or CDC50B-Recent studies have revealed that CDC50 proteins co-immunoprecipitate with class 1 P4-ATPases (ATP8A1, ATP8A2, ATP8B1, ATP8B2, ATP8B3, and ATP8B4) and are required for transport of these P4-ATPases from the ER (23)(24)(25)37). We first sought to determine whether other P4-ATPases require CDC50A and/or CDC50B for their translocation from the ER to specific cellular compartments.…”

“…In contrast, the Neo1p P4-ATPase does not associate with either Cdc50p or Lem3p (14); furthermore, unique among the five yeast P4-ATPases, deletion of the NEO1 gene alone is lethal (22). Recently, the specificity of interactions of human class 1 P4-ATPases (ATP8A1, ATP8A2, ATP8B1, ATP8B2, ATP8B3, and ATP8B4) with CDC50 proteins and the requirement of CDC50 proteins for the subcellular localization of the ATPases have been characterized (23)(24)(25)(26). However, the functional association with CDC50 proteins and subcellular localization of other classes of human P4-ATPases remain largely unknown.…”

Structure and mechanism of ATP-dependent phospholipid transporters

“…Topologically, this protein resembles a fusion of the β-and γ-subunits of mammalian Na + /K + -ATPases [112]. As in the case of the Na + /K + -ATPase β-subunit, the Cdc50 protein is required for proper folding and exit of the P-type ATPase from the ER [111,[103][104][105][106][107][108][109][110][111][112][113][114][115][116]. For the Na + /K + -ATPase, the γ-subunit is involved in the modification of the kinetic parameters of cation transport depending on tissue and environmental conditions [117][118][119].…”