Abstract
Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.
Highlights
Phosphatidylserine (PS),3 and phosphatidylethanolamine are concentrated in the cytoplasmic leaflet [1, 2]
CDC50 Proteins Are Not Co-immunoprecipitated with ATP9A and ATP9B—We investigated whether CDC50A and CDC50B physically interact with P4-ATPases, including ATP9A and ATP9B
We found that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins for their exit from the endoplasmic reticulum (ER) and localization to specific cellular compartments where they perform their functions (Fig. 2)
Summary
RT-PCR—Total RNA from HeLa and RPE1 cells was isolated using an RNeasy minikit (Qiagen) and subjected to RT-PCR analysis using a SuperScript III One-Step RT-PCR system (Invitrogen). Cloning—A full-length cDNA of ATP11C was obtained by amplification of HeLa cell total RNA by RT-PCR. Missing cDNA regions were obtained by amplification of HeLa total RNA and ligated to the partial cDNAs to construct full-length cDNAs. The full-length P4-ATPase cDNAs were cloned into the pENTR3C vector (Invitrogen). Human CDC50A was amplified by RT-PCR from HeLa total RNA and cloned into the pTAC1 vector by TA cloning (BioDynamics Laboratory). Cell Culture, siRNA-mediated Knockdown, and Immunofluorescence Analysis—Culture of HeLa cells and transfection of expression plasmids were performed as described previously [29, 30]. Immunoprecipitation—HeLa cells were transfected using FuGENE 6 or X-tremeGENE 9 (Roche Applied Science) with different combinations of expression vectors for P4-ATPase and CDC50 and grown for 2 days. Beads were incubated at 37 °C for 2 h in SDS sample buffer, and the supernatant was subjected to immunoblot analysis using rat anti-HA and mouse anti-FLAG or mouse anti-TfnR antibodies
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