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 es-

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Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P 4 P-type adenosine triphosphatase (ATPase) family of putative aminophospholipid translocases, and loss of aminophospholipid asymmetry in the canalicular membranes of ATP8B1-deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain-specific aminophospholipid translocase activities and polarized organization of polarized epithelial Caco-2 cells. Caco-2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin-labeled phospholipids. The polarized organization of these cells was determined by pulse-chase analysis, cellfractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco-2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither aminophospholipid transport nor the asymmetrical distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1-depleted Caco-2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression. Conclusion: Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency.

Most plasma membranes display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is critical for cell survival in multicellular organisms but it's purpose for the functioning of individual cells is unclear. A general view is that membrane asymmetry is created by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip PS and PE from the external to the cytosolic leaflet. The identity of APLTs is not known but prime candidates are members of the P4 subfamily of P‐type ATPases. A systematic analysis of these proteins in budding yeast revealed that: P4 ATPases are essential for aminophospholipid transport and asymmetry; asymmetry is not created at the plasma membrane but rather when membrane flows through the Golgi; loss of P4 ATPase function perturbs vesicle formation in the late secretory and endocytic pathway; P4 ATPases interact with proteins implicated in coat recruitment. Our current work focuses on how P4 ATPases contribute to vesicle formation. As a first approach, we set out to purify and reconstitute P4 ATPases in giant liposomes to test whether these proteins can drive membrane vesiculation. As a complementary approach, we use a proteomics‐based strategy to identify P4 ATPase binding partners that control lipid transport in relation to other aspects of vesicle formation like coat recruitment and cargo sorting.

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