Flip or flop: functional analysis of a disease-related class of lipid pumps

Abstract

A fascinating aspect of cellular membranes is that the different lipid species are often non-randomly distributed across the bilayer. This lipid asymmetry serves a multitude of cellular functions and is maintained by uni-directional flippases. The identity of these activities remains to be established, but promising candidates are members of the P4 subfamily of P-type ATPases. Dysfunction of the latter enzymes has been implicated in diabetes, obesity and a life-threatening liver disease. Consequently, much attention is currently focused on the mechanisms underlying lipid asymmetry and their (patho)physiological implications. Mutation of the human P4 ATPase ATP8B1 causes the life-threatening liver disease familial intrahepatic cholestasis type 1 (FIC1 disease). Here, we explore the origin of the extrahepatic symptoms associated with FIC1 disease by investigating the impact of blocking ATP8B1 expression on the membrane domain specific flippase activities and functional organization of polarized human intestinal epithelial cells. Although ATP8B1 is abundantly expressed in the apical membrane, we show that blocking its expression by RNAi neither affects the apical flippase activity, nor the steady state distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1 depletion has a profound effect on apical membrane organization, including a dramatic loss in microvilli and down regulation of apical membrane protein expression. Importantly, these results point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed flippase activity, yet potentially relevant for the manifestation of FIC1 disease. Previous work indicates that aminophospholipid transport and asymmetry critically depend on P4 ATPases and their CDC50 binding partners. However, whether P4 ATPases directly catalyze aminophospholipid transport and whether or not CDC50 proteins are integral parts of the P4 ATPase transport machinery is not known. Hence, the molecular composition and working mechanism of aminophospholipid translocases remain to be established. Human P4 ATPase family members outnumber CDC50 homologues by 5 to 1. This implies that each human CDC50 protein interacts with multiple P4 ATPases or, alternatively, that some human P4 ATPases function without a CDC50 binding partner. To gain further insight into the significance of CDC50 proteins in P4 ATPase-dependent aminophospholipid transport, we systematically investigated the physical and functional interactions between members of these two protein families. We demonstrate that each human CDC50 homologue interacts with multiple P4 ATPases, and that in all cases analyzed these interactions are indispensable for P4 ATPase export from the ER. In addition, we provide the first evidence to date that CDC50 proteins serve a critical role in the reaction cycle of P4 ATPases. As CDC50 proteins seem to represent indispensable components of the P4 ATPase transport machinery, we next analyzed the consequences of a combinatorial loss of CDC50 proteins on the transbilayer organization of aminophospholipids in the plasma membrane of human cervical carcinoma cells. We show that blocking the expression of the two principal endogenous CDC50 proteins simultaneously did not lead to any measurable perturbation of aminophospholipid asymmetry at the plasma membrane. Experiments with CDC50 null mutants will be necessary to define the contribution of each CDC50 homologue to aminophospholipid transport and asymmetry