Biochemical and cellular functions of P4 ATPases - Biochemical Journal

Biochem. J. (2010) 431, 1–11 (Printed in Great Britain)

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doi:10.1042/BJ20100644

REVIEW ARTICLE

Biochemical and cellular functions of P4 ATPases Lieke M. VAN DER VELDEN, Stan F. J. VAN DE GRAAF and Leo W. J. KLOMP1 Department of Metabolic and Endocrine Diseases, UMC Utrecht, and Netherlands Metabolomics Centre, Utrecht, 3508 AB, The Netherlands

P4 ATPases (subfamily IV P-type ATPases) form a specialized subfamily of P-type ATPases and have been implicated in phospholipid translocation from the exoplasmic to the cytoplasmic leaflet of biological membranes. Pivotal roles of P4 ATPases have been demonstrated in eukaryotes, ranging from yeast, fungi and plants to mice and humans. P4 ATPases might exert their cellular functions by combining enzymatic phospholipid translocation activity with an enzyme-independent action. The latter could be involved in the timely recruitment

of proteins involved in cellular signalling, vesicle coat assembly and cytoskeleton regulation. In the present review, we outline the current knowledge of the biochemical and cellular functions of P4 ATPases in the eukaryotic membrane.

INTRODUCTION TO P4 ATPases

phospholipids, also called ‘flop’, from inner cytosolic to outer or luminal leaflet at the expense of ATP (reviewed in [5]). The third class of proteins that facilitate transverse transport of phospholipids across biological membranes are the APLTs (aminophospholipid translocases) or ‘flippases’ which are the subject of the present review. APLT activity was demonstrated in the human erythrocyte plasma membrane [6–8] and in membranes of bovine intracellular chromaffin granules, which was attributed to a protein named ATPase II [9]. The APLT activity in these studies is responsible for the translocation of PS and PE, but not PC, from the external (or luminal) leaflet of the plasma membrane to the inner (or cytosolic) leaflet in an ATP-dependent manner. Since the biochemical discovery of APLT activity, the molecular identity of the translocase remained elusive for over a decade until Tang et al. [10] purified the ATPase II protein from chromaffin granules, determined part of the amino acid sequence and used this information to clone the full-length bovine ATPase II cDNA. They discovered that this bovine protein is a member of a previously unrecognized subfamily of P-type ATPases, now known as P4 ATPases [11]. ATPase II-homologous genes, including DRS2 (deficient for ribosomal subunits 2), were identified in yeast and Tang et al. [10] then demonstrated that a Drs2 strain appeared markedly deficient in translocase activity of fluorescently labelled PS across the plasma membrane. Together, these data demonstrated for the first time that APLT activity is a highly conserved function of the P4 ATPase family. To date, many researchers have provided evidence for the important roles of this class of proteins in a plethora of organisms, ranging from plants, yeast and nematodes to humans [12–18].

Membrane lipid asymmetry and P4 ATPases

Biological membranes exist of amphipathic lipid molecules that self-assemble into bilayers in an aqueous environment due to their intrinsic property of a hydrophilic headgroup and a lipophilic tail. The main lipid species of cellular membranes in eukaryotic cells are PC (phosphatidylcholine), PS (phosphatidylserine), PE (phosphatidylethanolamine) and PI (phosphatidylinositol) which belong to the group of glycerophospholipids. Other major classes of lipids are SM (sphingomyelin) and glycerosphingolipids, which belong to the class of the sphingolipids [1]. Lipid bilayers of biological membranes display a striking asymmetric distribution of lipids, including phospholipids [2]. Along the secretory and endosomal pathways, there is a distribution gradient of phospholipids in a non-random asymmetrical manner. Phospholipids in the two hemileaflets of the ER (endoplasmic reticulum) membrane are symmetrically distributed, whereas the lipids in the Golgi and the plasma membrane adopt an asymmetrical distribution. PS, PE and PI are enriched at the cytosolic leaflet of the plasma membrane and internal organelles, whereas lipids such as SM, PC and glycosphingolipids face the luminal or outer leaflet. Whereas lipids can freely diffuse in the lateral plane of the membrane, transverse, or flip-flop, movement of lipids in the plasma membrane is very slow (t 12 =hours to days) [1]. For the creation and maintenance of the asymmetrical distribution of phospholipids, eukaryotic cells have evolved an elaborate set of proteins, which are divided into three classes. The first class of proteins are the phospholipid scramblases which facilitate a nonlipid-specific transverse movement of phospholipids in either direction [3,4]. The second class of proteins are the ABC (ATP-binding cassette) transporters or so-called ‘floppases’. This protein family facilitates the transbilayer movement of

Key words: aminophospholipid translocases, cell membrane, flippase, phospholipid, subfamily IV P-type ATPase (P4 ATPase).

Phenotypes associated with P4 ATPase dysfunction

Through knockdown and knockout experiments and by characterizing the effects of genetic mutations in P4 ATPases,

Abbreviations used: ABC, ATP-binding cassette; ALA, aminophospholipid ATPase; ALIS, ALA-interacting subunit; APLT, aminophospholipid translocase; Arf, ADP-ribosylation factor; Cdc, cell-division cycle; ER, endoplasmic reticulum; GEF, guanine-nucleotide-exchange factor; P4 ATPase, subfamily IV Ptype ATPase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PKC, protein kinase C; PS, phosphatidylserine; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase; SM, sphingomyelin; TAP, tandem affinity purification. 1 To whom correspondence should be addressed (email [email protected]).  c The Authors Journal compilation  c 2010 Biochemical Society

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L. M. van der Velden, S. F. J. van de Graaf and L. W. J. Klomp

aberrant P4 ATPase function has been studied in different organisms. In this section, we describe the various phenotypical consequences of P4 ATPase deficiencies. Consequences at the molecular and cellular level are described below. Lyssenko et al. [15] have systematically investigated expression and developmental function of the six P4 ATPase genes encoded in the Caenorhabditis elegans genome, called tat-1–tat-6. From these genes, tat-5 is the only ubiquitously expressed essential P4 ATPase. tat-1–tat-4 are individually dispensable, although they seem to be only partly redundant as they become essential when the nematodes are metabolically challenged [15]. Similar studies have been performed in yeast. From the five P4 ATPase genes, only NEO1 (the name refers to its identification in a screen for genes that confer resistance to the aminoglycoside neomycin) was reported to be an essential gene [19]. The quadruple mutant lacking the four other yeast P4 ATPases is unviable, although any one member of this group can maintain viability, indicating that there is a functional overlap between the encoded proteins [20]. However, evidence is accumulating that some of the P4 ATPases also perform more specialized functions (see below). Redundancy among the P4 ATPases is often partial and depends on the readout, making studies on the function(s) of these proteins difficult and hard to interpret. The requirement of P4 ATPases is determined under various challenging conditions, including the presence of antibiotics, heavy metals and cytolytic chemicals [17,19,21]. Furthermore, several screens have been performed that show the essential role of specific P4 ATPases when other genes are knocked out (synthetic lethality) (see below). Although the underlying mechanisms are not always understood, these studies have elucidated pathways in which P4 ATPases are involved (see below and Figure 2). Below we discuss the distinct roles of selected P4 ATPases in organisms grown at reduced temperature, in polarized growth and describe P4 ATPase-related phenotypes in mice and humans.

Cold-sensitivity

Drs2 cells exhibit a cold-sensitive growth phenotype [13,19,20]. These strains are viable at temperatures above 23 ◦C, but fail to grow at temperatures below 18 ◦C. Similarly, Dnf1Dnf2 double-mutant yeast cells show a cold-sensitive growth defect in the biogenesis of endocytic vesicles [17]. Drs2 mutants were also discovered in a screen for genes that were essential under highpressure conditions [22]. Abe and Minegishi [22] suggest that the cold and high-pressure growth defects of Drs2 mutants are due to severe intracellular trafficking defects. Interestingly, coldsensitive growth defects related to aberrant P4 ATPase function have also been observed in higher eukaryotes. Arabidopsis thaliana with an ALA1 (aminophospholipid ATPase 1) mutation had a significantly reduced size when grown in a cold environment compared with wild-type plants. Furthermore, transformation of ALA1 into yeast Drs2 mutants rescued the cold-sensitive phenotype of these mutants [23]. Cold-sensitivity was also observed with a newly identified mouse P4 ATPase FetA [24]. Xu et al. [24] identified FetA as a specific testis-expressed P4 ATPase with 43 % amino acid identity with Atp8b3, which is also a specific testis-expressed P4 ATPase in mouse and humans. FetA localized at the acrosomal ridge which is a specialized membranous region derived from the Golgi in spermatocytes [24]. RNAi (RNA interference)-mediated knockdown of FetA in mouse P815 cells resulted in abnormal Golgi structures when cells were cultured at 33 ◦C, but not at 37 ◦C [24]. These observations suggest that several P4 ATPases have specific essential functions.  c The Authors Journal compilation  c 2010 Biochemical Society

Polarized growth

In yeast, growth of the bud tip is a tightly regulated process. During normal bud tip growth, PE becomes temporarily exposed in the outer leaflet of the bud tip membrane. Both Dnf1p and Dnf2p localize at the sites of polarized growth of the bud tip and are responsible for PE flip at the bud tip site [17,20,25,26]. Saito et al. [27] described that the effect of PE flippase activity in polarized growth is mediated by Cdc (cell-division cycle) 42 [27], a known regulator of polarized bud growth in Saccharomyces cerevisiae [28]. Cdc42 is recruited to the site of bud growth by a Ras family GTPase and is activated there by its GEF (guaninenucleotide-exchange factor), Cdc24. Activated Cdc42–GTP leads to rearrangements of the actin cytoskeleton and to growth of the bud tip by polarized delivery of cellular components. Cdc42–GTP hydrolysis is regulated by three GTPase-activating proteins, which are in turn regulated by PE and PS in the inner membrane leaflet. Consequently, a functional Dnf1/Dnf2 mutant shows prolonged apical bud growth [27]. The involvement of P4 ATPases in polarized growth has also been demonstrated in other unicellular organisms such as the rice plant pathogenic fungus Magnaporthe grisea. The fungus P4 ATPases MgPDE1 and MgAPT1 play an important role in the polarized growth of the hyphal appressoria and in the formation of exocytic vesicles respectively [29,30]. Also, in multicellular eukaryotes, P4 ATPase dysfunction can manifest as aberrant polarized growth. Mutations in the A. thaliana ALA3 gene resulted in reduced pollen tube growth, reduced primary root growth and longer root hairs. Mutations in ALA3 also resulted in abnormal morphology of trichomes, which are plant epidermal outgrowths [31]. P4 ATPase-associated diseases in humans and mice

The human P4 ATPases ATP8A2, ATP11A and ATP11B have been anecdotally reported to be involved in tumorigenesis or poor outcome of lung transplantation patients [32–34]. However, direct involvement of these P4 ATPases to disease-related processes remains to be demonstrated. To date, only one of the 14 human P4 ATPases has been unequivocally related to a disease. Mutations in the ATP8B1 gene cause a hereditary cholestasis syndrome which is characterized by an impairment of bile flow from the liver and various extrahepatic manifestations, including diarrhoea and hearing loss [12,35]. ATP8B1 is expressed in apical membranes of polarized epithelial cells, including hepatocytes, pancreatic acinar cells, intestinal enterocytes and cochlear hair cells [36– 38]. Cellular depletion of ATP8B1 leads to severe alterations in the structure of the apical membrane, with degeneration or even loss of microvilli in enterocytes and hepatocytes, and progressive loss of stereocillia in the outer hair cells of the cochlea, which can explain the symptoms in patients with ATP8B1 deficiency [36,38–41]. See also below in the ‘P4 ATPases and membrane stability’ section. In mice, additional phenotypes related to aberrant function of P4 ATPase family members have been described. Mouse Atp8b3 is localized in the acrosomal region of sperm cells. Sperm cells of Atp8b3−/− mice have increased PS exposure in the outer leaflet and are unable to release digestive enzymes and penetrate the zona pellucida. This is reflected in the smaller litter sizes of Atp8b3−/− male mice [42,43]. In mice, Atp10a (also referred to as Atp10c) is an imprinted gene which shows strong genetic association with fat and insulin metabolism and the regulated uptake of glucose in adipose tissue and skeletal muscle [44]. Like Atp10a, Atp10d is associated with obesity in mice [45]. The C57BL/6 mouse strain caries a stop

Biochemical and cellular functions of P4 ATPases

codon in exon 12 of Atp10d. The C57BL6 mice are known to be prone to obesity when fed on a high-fat/high-glucose diet. Genetic linkage studies may help to clarify whether ATP10A or ATP10D also represent susceptibility genes for obesity in humans. There are a few reports that link the chromosomal region 15q11-q13, in which ATP10A resides, and the imprinting status of this region to autism spectrum disorders [46–49]. STRUCTURAL ASPECTS OF P4 ATPASES

Although no structural information is available for P4 ATPases, some insight into their working mechanism can be gained from data on other P-type ATPases [50,51]. P-type ATPases are highly specialized transporters that translocate a specific substrate across a membrane at the cost of ATP hydrolysis [52]. Hence the name P-type ATPase in which the P stands for the transient phosphorylation of a conserved aspartate residue (D in DKTG) during the catalytic cycle. The description of the pumping cycle is known as the Post–Albers cycle [53,54], and the crystal structures of several P-type ATPases in various stages of the Post– Albers cycle have been solved over the years. Despite significant sequence differences between these proteins, their structure is remarkably similar [55]. Lenoir et al. [50,56] discussed this P-type transport cycle in relation to the role of P4 ATPases in the translocation of phospholipids from the exoplasmic to the cytosolic leaflet. They suggest that the phospholipid ligand in P4 ATPases binds to the E2-P configuration, in contrast with Ca2+ in the P-type Ca2+ -pump SERCA (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase), which binds to E1. This is in line with the observation that the P4 ATPase ATP8A1 rapidly dephosphorylates upon PS addition [57], whereas SERCA is phosphorylated upon addition of Ca2+ . The ligand-binding domains of cation-transporting P-type ATPases comprise negatively charged amino acid residues in transmembrane helices 4 and 6. Interestingly, P4 ATPases have neutral amino acids at these positions [10]. This could reflect an evolutionary adaptation to transport phospholipids instead of cations, although it remains unclear where P4 ATPases fit the bulky lipid and the headgroup translocation pathway. Answering this question will probably require successful crystallization and structure elucidation of a P4 ATPase, which has not been achieved to date. THE P4 ATPase–Cdc50 HETEROMERIC COMPLEX

One important conserved feature of P4 ATPases across species is that they form heterodimers with members of the Cdc50 protein family [18,26,58–62]. The CDC50 gene was originally identified in a genetic screen for yeast mutants exhibiting cold-sensitive growth defects (similar to the phenotype of P4 ATPase-deficient yeast) [58,63,64]. These proteins have two transmembrane spanning helices, relatively short cytosolic N- and C-terminal tails, a large extracellular loop with one or more predicted internal disulfide bonds and multiple putative glycosylation sites [26,58] (Figure 1). The different eukaryotic genomes studied have no clear relationship between the number of P4 ATPases and the number of CDC50 genes present, except that all genomes contain more P4 ATPases than CDC50 genes (Table 1). This suggests that Cdc50 proteins may associate with more than one P4 ATPase or that there are P4 ATPases which operate on their own. Indeed, the S. cerevisiae and A. thaliana Cdc50 proteins Lem3p, ALIS (ALA-interacting subunit) 1, ALIS3 and ALIS5 functionally

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Table 1 Overview of number of P4 ATPase genes and Cdc50 family member genes identified in different eukaryotic genomes One P4 ATPase gene and one CDC50 gene were identified in L. donovani , but the genome of this organism was not analysed for additional P4 ATPase and CDC50 genes. Species

P4 ATPase

Cdc50

Reference(s)

Homo sapiens Mus musculus Caenorhabditis elegans Saccharomyces cerevisiae Arabidopsis thaliana Drosophila melanogaster Magnaporthe grisea Leishmania donovani

14 14/15 6 5 12 6 4 1

2/3 3 ? 3 5 ? ? 1

[120–122] [24,121] [120,123] [74,124] [18,23,29] [123] [30] [16,61]

Figure 1

Schematic representation of a P4 ATPase and its Cdc50 subunit

P4 ATPases contain ten transmembrane helices. Cdc50 proteins have short cytosolic N- and C-termini, multiple putative glycosylation sites (represented by the branched structures) and one or more putative internal disulfide bridges (not shown).

associate with (at least) two P4 ATPases [25,26,65]. Furthermore, some P4 ATPases appear to interact with more than one Cdc50 protein. Human ATP8B1 functionally interacts with both Cdc50A and Cdc50B [60] and the plant P4 ATPases ALA2 and ALA3 functionally interact with three different plant Cdc50 proteins [18,65]. The exact function of Cdc50 proteins in relation to P4 ATPases is not known. Cdc50 proteins may act as a folding chaperone, as a determinant of the subcellular localization, as a determinant of substrate specificity, as an essential catalytic subunit of the P4 ATPase–Cdc50 complex, or a combination of these possibilities. A chaperone function of Cdc50 proteins in relation to P4 ATPases has indeed been demonstrated in different organisms. P4 ATPases need to associate with a Cdc50 family member to exit the ER [18,26,58–62]. Cdc50 proteins also require association with a P4 ATPase for ER exit, complex glycosylation and plasma membrane localization [62,65]. A specific role for Cdc50 proteins as a determinant of P4 ATPase subcellular localization beyond ER-exit has not been equivocally demonstrated. Recently, Lopez-Marques et al. [65] investigated two A. thaliana P4 ATPases, ALA2 and ALA3 in relation to three A. thaliana Cdc50 family members, ALIS1, ALIS3 and ALIS5. They interrogated whether distinct Cdc50 family members determine the subcellular localization and substrate specificity of the P4 ATPases with which they associate. Their data clearly indicate that subcellular localization and substrate preference are specified by molecular signals within the P4 ATPase and not by the Cdc50 protein [65]. Recent work by Lenoir et al. [56] elegantly indicates that S. cerevisiae Cdc50 proteins are crucial components of the catalytic cycle of the P4 ATPase  c The Authors Journal compilation  c 2010 Biochemical Society

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L. M. van der Velden, S. F. J. van de Graaf and L. W. J. Klomp

complex. They purified the yeast P4 ATPase Drs2p to apparent homogeneity in the presence or absence of Cdc50p. These experiments revealed that the association of Drs2p with Cdc50p was required for phosphorylation of aspartate residues. They also provided indirect evidence that the Cdc50p–Drs2p interaction is dynamic during the reaction cycle and that this interaction is strongest when Drs2p is in the E2P conformation. On the basis of this work, Puts and Holthuis [51] postulated that Cdc50 protein function in relation to P4 ATPases is analogous to the function of the β subunits of P2 ATPases. It has been well established that the α and β subunits of P2 ATPases together form a catalytically active complex, whereas the individual subunits have no catalytic activity (reviewed in [66]). The crystal structures of several P2 ATPases in complex with their respective β subunits have recently been solved, and indicate that β subunits are important for the stabilization of the α subunit during cation transport [67,68].

FUNCTIONS OF P4 ATPases Biochemical functions Aminophosphospholipid translocase

After the seminal paper by Tang et al. [10] providing genetic evidence that P4 ATPases were the prime candidates for the longsought APLT [10], a large number of studies investigating the role of P4 ATPases have been published. Initially, most studies focused on yeast P4 ATPases. Bull et al. [12] were the first to link genetic mutations in a P4 ATPase to an inherited disease in humans. Many additional studies linked yeast P4 ATPase activity to maintenance of membrane phospholipid asymmetry in yeast and in other organisms [18,21,26,59,60,69–71]. In the literature, several inconsistencies exist regarding the APLT function of Drs2p [72,73]. These can mainly be ascribed to the fact that the yeast genome contains five P4 ATPase genes with largely redundant functions [17,20,74]. Next, deletion of DRS2 has also consequences on the trafficking of two P4 ATPases, Dnf1p and Dnf2p, that were primarily responsible for maintaining phospholipid asymmetry at the plasma membrane [17,75]. In addition, it appeared that Drs2p is a P4 ATPase that recycles between the Golgi apparatus and the endocytic vesicular pathway, thus predominantly establishing phospholipid asymmetry in intracellular membranes [76], whereas initial studies were directed mainly towards measuring APLT activity at the plasma membrane [10,72,73]. The interpretation of genetic studies on P4 ATPase function is hampered by the notion that disruption of the expression of one specific P4 ATPase in many cases caused intracellular vesicular trafficking defects, thus potentially prohibiting the proper localization and function of other P4 ATPases or even other proteins that could possess APLT activity [13,14,17,20,76]. Ding et al. [57] and Paterson et al. [77] isolated overexpressed bovine and murine Atp8a1 from insect cells and showed that the ATPase activity of the enzymes was specifically enhanced when PS was added to the reaction mixture. This indicated that PS is an endogenous substrate of Atp8a1. Definitive proof that P4 ATPases are APLTs was recently provided by Zhou and Graham [78]. They purified and reconstituted the yeast P4 ATPase Drs2p into proteoliposomes, resulting in lipid translocation of fluorescently labelled PS [78]. Similarly, Coleman et al. [79] purified and reconstituted bovine Atp8a2 from retina photoreceptor disc membranes and demonstrated lipid translocase activity which is specific for fluorescently labelled PS {NBD [N-(7-nitro-2,1,3-benzoxadiazol-4-yl)]-PS}. This work provided compelling evidence that these P4 ATPases are phospholipid translocases in vitro.  c The Authors Journal compilation  c 2010 Biochemical Society

Enzyme-independent function of P4 ATPase

In addition to the lipid translocase function, P4 ATPases probably also have a pump-independent function (Figure 2). Initial evidence for this centred on the cold-sensitivity of yeast mutants with defects in Drs2p. Deletion of DRS2 in S. cerevisiae causes a cold-sensitive phenotype. The cold-sensitive growth phenotype of Drs2 yeast cells could not be rescued by transformation with a Drs2p C-tail truncation mutant, although this mutant protein localized properly and was expected to have PS translocase activity. Similarly, cold-sensitive growth could not be rescued by expression of Drs2p-D560N, an ATPase-deficient mutant. However, when these two Drs2p mutants were transformed together into Drs2 yeast cells, growth at the permissive temperature was restored [80]. This suggests that Drs2p performs two independent and essential functions where the C-terminal truncation mutant provides the enzymatic transport function of Drs2p, whereas the enzymatic dead mutant complements the pump-independent function of the C-terminal tail of Drs2p. A second line of evidence for a pump-independent function of Drs2p came from Zhou and Graham [78], who purified Drs2p with a TAP (tandem affinity purification) tag at either the N-terminus or the C-terminus (TAP– Drs2p and Drs2p–TAP) and reconstituted these proteins in proteoliposomes. PS translocase activity was only observed in vitro with TAP–Drs2p, whereas Drs2p–TAP seemed catalytically dead. Interestingly, both N- and C-terminally tagged DRS2 constructs were able to rescue the cold-sensitive growth defect of Drs2 cells [78]. This suggests that these enzymes have a pump-independent function in addition to their APLT activity, and that the former is important for rescuing the cold-sensitive growth phenotype. The pump-independent function is not only reflected in a coldsensitive phenotype and is not restricted to yeast, as demonstrated by two groups. First, Natarajan et al. [76] showed that trans-Golgi membranes contain Drs2p-dependent PS translocase activity and Drs2 yeast mutants show profound protein and vesicular trafficking defects. They tested whether the translocation of the substrate PS (the enzymatic function of Drs2p) was necessary for proper intracellular vesicular traffic. However, the mere absence of (translocated) PS did not explain the trafficking defects in Drs2 cells. Deletion of the PS-synthase gene CHO1 completely eliminates PS synthesis, rendering this strain devoid of PS. Remarkably, CHO1 mutants are viable and grow reasonably well on rich medium, but do not show protein transport defects like drs2 mutants. This suggested that Drs2p performs two separate functions: the enzymatic translocation of PS and, in addition, a PSindependent function which is involved in the vesicular trafficking phenotype. The latter implies either that PS is not an essential substrate for Drs2p-mediated vesicle formation or that Drs2p has a translocase-independent function. Secondly, knockdown of the mammalian P4 ATPase ATP8B1 in polarized human intestinal epithelial (Caco-2) cells in vitro did not affect the internalization rate of spin-labelled PS, but severely affected the organizational structure of the apical plasma membrane [38], again suggesting that a P4 ATPase, ATP8B1, might perform a pump-independent function. In analogy to the study of Chantalat et al. [80] in yeast, attempts were made to rescue this phenotype using ATP8B1 mutant proteins either lacking the C-terminus or catalytically dead (Asp454 mutants). Unfortunately, these were unsuccessful due to the fact that these mutations resulted in ATP8B1 misfolding and ER-retention ([62] and L.M. van der Velden and L.W.J. Klomp, unpublished work). Interestingly, non-pumping functions have also been described for other P-type ATPases. Several studies highlight the ATPaseindependent role of the Na+ /K+ -ATPase as a signal transducer [81–83]. Also, a pool of non-pumping Na+ /K+ -ATPase has been

Biochemical and cellular functions of P4 ATPases

Figure 2

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Biochemical and cellular functions of P4 ATPases

Simplified schematic representation of P4 ATPase-dependent functions in a (polarized) eukaryotic cell. Biochemical and cellular functions of P4 ATPases addressed in the present review are indicated by seven numbers in the P4 ATPase-deficient cell. The first two comprise pump-dependent functions. (1) APLT activity of P4 ATPases. (2) P4 ATPases potentially influence signal transduction cascades by controlling availability of PE and PS in space and time. (3) Cold-sensitivity is a pump-independent function of yeast and plant P4 ATPases, but it is currently unclear which cellular process is responsible for this phenotype. Finally, four distinct phenotypes of P4 ATPase-deficient cells (labelled 4–7) cannot be solely attributed to the APLT activity or pump-independent function. (4) Reduced assembly/functioning of ribosomes is observed in P4 ATPase-deficient cells. (5) Malfunction of exocytic and/or endolysosomal trafficking is observed in P4 ATPase-deficient cells. (6) Loss of membrane stability/microvilli or polarized growth. Loss of microvilli in ATP8B1-deficient cells is currently mainly attributed to the pump-independent function of this protein. (7) Sterol import is decreased in P4 ATPase-deficient cells. Numbers on a dark-grey background represent P4 ATPase APLT function, numbers on a white background represent P4 ATPase pump-independent functions and numbers on a light-grey background represent processes for which it is at present unclear whether the APLT activity, the pump-independent function or both functions are required.

described [84]. The Na+ /K+ -ATPase recruits several proteins by direct interaction with the α subunit, thus effectively functioning as a scaffold for signalling proteins [81–83,85]. Binding of cardiac glycosides such as ouabain to Na+ /K+ -ATPase in this receptor complex induces cellular signalling cascades eventually resulting in changes in the expression of a number of growth-related genes and in cell proliferation [81–83]. Considering the large number of protein–protein interactions described for P4 ATPases in yeast, a similar pump-independent scaffold function of these proteins can easily be envisioned (Table 2 and see the ‘P4 ATPases and vesicular trafficking’ section below). Cellular functions of P4 ATPases

The distinct biochemical functions of P4 ATPase as described above are implicated in a variety of cellular processes. Below, we summarize the cell biological functions of P4 ATPases and discuss whether this function can be attributed to the molecular function of P4 ATPase, either to the flippase function or to the putative pump-independent scaffold function or both. The biochemical and cellular functions of P4 ATPases are represented schematically in Figure 2. P4 ATPases and signal transduction at the cytosolic leaflet

In addition to their prime function in establishing organelle and cell boundaries by forming lipid bilayers, lipids also function as

signalling molecules [86]. Well-known examples of lipids that serve as signalling molecules are the phosphoinositides and DAG (diacylglycerol). Lipids can recruit, activate or inhibit protein activity or a combination of these processes at a membrane (reviewed in [86–88]). Yeung et al. [89] created a fluorescent PS probe based on the PS-binding domain of lactadherin, a glycoprotein of milk fused to GFP (green fluorescent protein). In mammalian cells, this probe localized to the plasma membrane, but also to vesicular structures which were identified as endocytic vesicles. They demonstrate that the association of positively charged proteins with (negatively charged) PS dynamically regulates the localization of these proteins. Furthermore, they suggest that changes in phospholipid distribution or metabolism, influx of positive ions such as Ca2+ or phosphorylation of proteins can relocalize the proteins and thus the reactions catalysed by them [89,90]. An example of a PS-binding protein is PKC (protein kinase C) (for other examples, see [88]). Almost all PKC isoenzymes need PS as a cofactor for activation. By inference, altered spatial or temporal distribution of PS due to P4 ATPase activity could potentially lead to reduced PKC activation. This hypothesis was tested directly in UPS-1 cells, a mutant CHO (Chinese-hamster ovary) cell line characterized by an unknown endogenous defect leading to reduced PS-flippase activity [91]. Overexpression of ATP8B1 in UPS-1 cells resulted in induction of PS translocation [60] with a concomitant activation of endogenously expressed PKCζ [92]. Therefore dysfunctional P4 ATPases can potentially influence the spatial and or temporal  c The Authors Journal compilation  c 2010 Biochemical Society

6 Table 2

L. M. van der Velden, S. F. J. van de Graaf and L. W. J. Klomp Overview of P4 ATPase and/or Cdc50 interactions

AP-1, adaptor protein 1; GAP, GTPase-activating protein; mmBCFA, monomethyl branched-chain fatty acid; SNARE, soluble N -ethylmaleimide-sensitive fusion protein-attachment protein receptor. P4 ATPase/Cdc50 family member

Interaction partner

Protein function of interaction partner

Type of interaction

Reference

DRS2 NEO1 CDC50 /LEM3 /CRF1 DRS2 DRS2 NEO1 CDC50-DRS2 DRS2 DRS2 NEO1 DRS2 DRS2 DRS2 DRS2 CDC50 CDC50 CDC50 CDC50 DRS2 DRS2 DRS2-npw1,2 mutant DRS2 CDC50 CDC50 CDC50 CDC50 CDC50 CDC50 CDC50 CDC50 CDC50 DRS2 DRS2 DRS2 DRS2 DRS2 tat-2 Drs2p Neo1p Cdc50p-Drs2p Drs2p Drs2p Drs2p Drs2p Drs2p Drs2p Drs2p

TEF3 YSL2 YPT32 SLA2 SEC1-1 ARL1 RCY1 SLA1 APL4 GGA2 ARF1 CHC1-ts PAN1-20 PIK1 GCS1 ARF1 CHC1-521 GGA1,GGA2 GCS1 PAN1-20 PAN1-20 GGA1,GGA2 ERG3 RGP1 VPS1 SRV2 RIC1 YPT6 ERG6 ERG2 ERG5 ERG6 ERG2 ERG5 ERG3 KES1 elo-5 Gea2p Ysl2p Rcy1p Sla1p Apl4p Apl2p Sac1p Ino1p Itr1p Tcb3p

Yeast homologue of translation elongation factor EF-1γ Arl1p GEF Rab family small GTPase Mammalian homologue is Hip1R which binds both to actin and clathrin Stimulates SNARE-dependent membrane fusion Arf-like protein F-box protein which is involved in recycling out of early endosomes independent of ubiquitination Cytoskeletal binding protein required for the assembly of cortical actin cytoskeleton AP-1γ subunit Golgi-localizing γ -adaptin Arf1 Clathrin heavy chain Eps15-related protein that interacts with yeast homologue of clathrin assembly protein AP180 Phosphoinositide 4-kinase Arf GAP Arf1 Clathrin heavy chain Golgi-localizing γ -adaptin Arf GAP Plays a role in actin-driven endocytosis Plays a role in actin-driven endocytosis Golgi-localizing γ -adaptin Ergosterol biosynthetic pathway Ypt6 GEF Yeast dynamin Component of cortical actin patches Ypt6 GEF Ssmall GTPase Ergosterol biosynthetic pathway Ergosterol biosynthetic pathway Ergosterol biosynthetic pathway Ergosterol biosynthetic pathway Ergosterol biosynthetic pathway Ergosterol biosynthetic pathway Ergosterol biosynthetic pathway Oxysterol-binding protein homologue 4 Enzyme necessary for the production of mmBCFAs Arf GEF Arl1 GEF F-box protein which is involved in recycling out of early endosomes independent of ubiquitination Cytoskeletal binding protein required for the assembly of cortical actin cytoskeleton AP-1γ subunit AP-1β subunit Phosphoinositide-4-phosphate phosphatase Inositol-1-phosphate synthase Myo -inositol transporter Synaptotagmin orthologue

Genetic, high-copy suppressor Genetic, high-copy suppressor Genetic, high-copy suppressor Epistatic Epistatic Genetic Genetic Genetic Genetic Genetic Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, synthetic lethal Genetic, extragenic suppressor Genetic, extragenic suppressor Protein–protein Protein–protein Protein–protein Protein–protein Protein–protein Protein–protein Protein–protein Protein–protein Protein–protein Protein–protein

[93] [102] [25] [14] [14] [102] [25] [125] [126] [127] [21] [21] [21] [103] [128] [128] [128] [128] [129] [125] [125] [126] [111] [111] [111] [111] [111] [111] [111] [111] [111] [111] [111] [111] [111] [112] [130] [13] [102] [25] [125] [126] [126] [131] [131] [131] [131]

availability of phospholipids and thus the distribution and activity of proteins (Figure 2, point 2). P4 ATPases and ribosome function

The yeast P4 ATPase DRS2 gene was first identified in a functional screen for ribosome assembly mutants, hence the name Drs2, deficient for ribosomal subunits 2 [93]. Drs2 cells grown at the non-permissive temperature displayed delayed processing of 20S rRNA precursor into 18S rRNA (Figure 2, point 4). Interestingly, overexpression of a yeast translation elongation factor (TEF3) suppressed the cold-sensitive growth phenotype of Drs2 [93] (Table 2). A second P4 ATPase identified in yeast, NEO1, was discovered in a screen for resistance to aminoglycosides, which  c The Authors Journal compilation  c 2010 Biochemical Society

has also been implicated in ribosomal function [19]. Recently, a marked reduction in translation efficiency of alkaline phosphatase mRNA was observed in ATP8B1-depleted human intestinal cells (Figure 2, point 4) [38]. These results suggest that mammalian P4 ATPases might also have a connection in ribosome function. In yeast, ribosome biogenesis defects have also been observed in mutants that perturb the secretory pathway [94]. Therefore reduced translation efficiency could be a secondary effect of eukaryotic cells due to defects in vesicular trafficking. P4 ATPases and vesicular trafficking

Biogenesis of endocytic and secretory vesicles requires dynamic regulation of transbilayer lipid movement. P4 ATPases in S.

Biochemical and cellular functions of P4 ATPases

cerevisiae reside in different organelles of the exocytic pathway and have a crucial role in the formation of plasma membrane and Golgi-derived transport vesicles (reviewed in [75,95]). Interestingly, trafficking defects associated with aberrant P4 ATPase function are also observed in other organisms (Figure 2, point 5). As a first example, the P4 ATPase ALA3 of A. thaliana localizes at the Golgi and is expressed in root tip peripheral columella cells. This cell type produces a special kind of slime vesicle which is almost absent from ala3 mutant plants [18]. Secondly, MgAPT2 (M. grisea aminophosphlipid translocase 2) was identified as a homologue of the P4 ATPase S. cerevisiae DRS2, in the rice plant pathogenic fungus M. grisea. It was shown that the Golgi plays an important role in the penetration and infection potential/mechanism of M. grisea. Mgapt2 mutants show a decreased ability to infect host plants, show a decrease in the secretion of extracellular enzymes and accumulate abnormal Golgi-like cisternae [30]. As a third example, the C. elegans P4 ATPase Tat-1 is expressed in epidermal and intestinal cells. Tat-1 mutant nematodes accumulate abnormal intestinal vacuoles and early endosomal markers are distributed abnormally. Furthermore, Tat-1 is involved in the formation of endolysosomal vesicles [70,96]. It was proposed that P4 ATPases contribute to the formation of exocytic and endocytic vesicles by transporting phospholipids from the outer (or luminal) leaflet to the cytosolic leaflet, which creates an imbalance in the phospholipid surface area between the two leaflets of the bilayer [97]. According to the coupled bilayer hypothesis of Sheetz and Singer [98], an excess of lipid on one side causes the lipid bilayer to bend, and this process then contributes to the formation of transport vesicles (extensively reviewed in [99,100]). Although APLT activity may enhance vesicle formation [97], this activity alone is not enough to drive this process. Coat proteins that assemble on vesicle buds of the ER, Golgi, plasma membrane or endolysosomal system are necessary to stabilize these membrane structures. The small GTPbinding protein Arf (ADP-ribosylation factor) and its effector proteins regulate the assembly of coat proteins (reviewed in [101]). Interestingly, DRS2 was identified as synthetic lethal in combination with Arf , providing the first report on a P4 ATPase involved in vesicular traffic [13]. Deletion of DRS2 was also lethal in combination with clathrin heavy chain mutants. These observations suggest that these gene products operate in the same or parallel pathways [13]. Consistent with this notion, Drs2p is localized at the trans-Golgi, and Drs2 cells display swollen Golgi cisternae and trafficking defects mainly related to the function of the trans-Golgi network [13,14,20,76]. Since then, a large number of genetic and biochemical interaction studies made it evident that Drs2p function is linked to formation of clathrin-coated vesicles in intracellular trafficking pathways and that the yeast P4 ATPase family members Dnf1p, Dnf2p (which are localized at the plasma membrane) and Dnf3p (which localizes at the Golgi) share redundant functions with Drs2p in APLT and in vesicular trafficking (Table 2) (reviewed in [75,95]). It is striking that P4 ATPases interact directly with several proteins involved in the regulation of vesicular transport, especially those that are involved in the recruitment of clathrin (Table 2), and thus function as scaffold factors for the generation of coats. Together, all of these data might be consistent with a hypothetical model that translocation of phospholipids is spatially and temporally coupled to coat recruitment by P4 ATPases and that this process substantially contributes to the formation of intracellular vesicles [75,102]. Interestingly, a number of recent studies link phosphoinositide signalling to P4 ATPase function. Sciorra et al. [103] identified DRS2 as a synthetic lethal gene with a temperature-sensitive

7

allele of PIK1, encoding a yeast phosphoinositide 4-kinase. Natarajan et al. [104] functionally characterized this interaction. A pik1ts mutant displayed reduced Drs2p-dependent phospholipid translocase activity in trans-Golgi membranes, which suggests that the presence of PtdIns4P is necessary for Drs2p activity [104]. Indeed, a PtdIns4P-binding region in the Drs2p C-terminus was identified. This binding region partially overlaps with an Arf GEF (Gea2p)-binding site, and binding of Gea2p also simulated Drs2p flippase activity [80]. Natarajan et al. [104] therefore suggest that these interactions of Drs2p with PtdIns4P and an Arf GEF serve as a coincidence detection system to spatially and temporally activate lipid translocation at the site of vesicle formation [104]. In addition, Puts et al. [131] identified three proteins involved in phosphoinositide metabolism as interactors of P4 ATPases: the inositol transporter (Itr1p), the phosphoinositide 4phosphatase (Sac1p) and the myo-inositol-1-phosphate synthase (Ino1p). Together with the studies of Nakano et al. [105] and Roelants et al. [106], these studies provide the first evidence that P4 ATPases are subject to regulation by an intricate network of protein kinases and signalling lipids, which together fine-tune P4 ATPase function.

P4 ATPases and membrane stability

One of the most astonishing effects of lipid translocation across a lipid bilayer is shape change. Shape change of the bilayer can either be local and result in vesiculation or tubulation, or affect the overall shape of the cell (recently reviewed in [100]). Cell shape is determined by bending of the lipid bilayer in combination with cytoskeletal properties. The first studies on the yeast P4 ATPases in this context involved structural analysis of membranes by electron microscopy [13,14,20,102]. This revealed the existence of aberrant intracellular membranes in Drs2, Neo1 and Drs2Dnf1 cells. These observations suggested that P4 ATPases play an important role in stabilizing membranes, most possibly by balancing phospholipids across membrane bilayer leaflets. Other clues that P4 ATPases may fulfil a crucial role in maintaining membrane stability came from studies investigating the mammalian P4 ATPase ATP8B1. Mutations in the ATP8B1 gene cause a hereditary cholestasis syndrome in humans [107]. Five studies by four different groups suggest that ATP8B1 plays a crucial role in maintaining stability of the apical plasma membrane of polarized epithelial cells (Figure 2, point 6). Bull et al. [108] used electron microscopy to study liver biopsies from ATP8B1-deficient patients. These biopsies showed ‘coarse granular bile’, shortened hepatocyte microvilli, filamentous packaging and disruption of the bile canalicular membrane. This observation was confirmed by a study of Paulusma et al. [41], who showed that Atp8b1 mutant mice have dilated bile canaliculi and shortened microvilli on the apical site of the hepatocyte. ATP8B1 specifically localizes at the apical plasma membrane of polarized cell types such as hepatocytes, cholangiocytes, enterocytes, acinar cells and cochlear hair cells [37,38,71,109]. Reduced expression of ATP8B1 or expression of an ATP8B1 mutant protein resulted in profound loss of microvillus(-like) structures in enterocytes and cochlear hair cells [36,38,39]. We propose that ATP8B1 plays an important function in stabilizing microvilli and ciliated structures at the apical plasma membrane. How exactly ATP8B1 stabilizes the apical plasma membrane is currently unknown, but could involve phospholipid translocation as well as recruitment of proteins involved in cytoskeleton dynamics. In erythrocytes, loss of phospholipid asymmetry also leads to profound morphological aberrations called echinocytic shape and is accompanied by  c The Authors Journal compilation  c 2010 Biochemical Society

8

L. M. van der Velden, S. F. J. van de Graaf and L. W. J. Klomp

disattachment of cytoskeletal elements from the membrane [110]. P4 ATPases and sterol homoeostasis

Studies in S. cerevisiae and C. elegans suggest that specific members of the P4 ATPase family play an important role in cellular cholesterol homeostasis (Figure 2, point 7). C. elegans expresses six P4 ATPases, and mutations in two of these results in a growth-arrest phenotype in a sterol-deprived environment [15]. In S. cerevisiae, knockout of one of ERG3, ERG2, ERG5 or ERG6, which are all ergosterol (yeast equivalent of cholesterol) biosynthesis genes appeared synthetic lethal with deletion of the P4 ATPase DRS2 and CDC50 [111] (Table 2), whereas single gene knockouts were all viable. This interaction seemed to be specific for the Drs2p–Cdc50p complex because double knockout of LEM3 and one of the ergosterol biosynthesis genes was not lethal. Furthermore, DRS2-deficient cells are hypersensitive to mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of ergosterol synthesis [112]. These results suggest that specific P4 ATPases are involved directly or indirectly in sterol import in S. cerevisiae and C. elegans. The involvement of only a specific P4 ATPase subset in these organisms suggests specialized functions in relation to sterols among the P4 ATPase family. A second connection between cholesterol and P4 ATPases was described by Muthusamy et al. [112]. They discovered that oxysterol-binding protein homologue 4 (Kes1 in yeast) antagonizes Drs2p-mediated flippase activity at trans-Golgi membranes (Table 2). Kes1p transfers oxysterols between membranes in vitro [113]. Conversely, Drs2p also seems to antagonize Kes1p activity, because Kes1p is hyperactive in Drs2 cells. This study suggests that Drs2p plays an important role in the trafficking of ergosterol from the membrane to intracellular sites. A third connection between sterol homoeostasis and P4 ATPase was demonstrated by Groen et al. [114] who observed ABCG5/ABCG8-independent cholesterol extraction from the apical plasma membrane of hepatocytes in ATP8B1-deficient mice [114]. They suggest that enhanced cholesterol escape from the outer leaflet is due to a disturbance in phospholipid asymmetry. It was proposed by Lange and Steck [115] that cholesterol ‘escapes’ more readily from the inner membrane leaflet than from the outer leaflet. Indeed, phospholipid scrambling increases cholesterol extraction from the outer membrane leaflet [116,117]. Taken together, the possibility exists that by regulating phospholipid asymmetry, P4 ATPases also control the availability of cholesterol at either site of the membrane. It is known that the amount of cholesterol in the membrane influences the activity of transmembrane transporters [118]. Therefore P4 ATPases potentially control the activity of other transmembrane transporters by indirectly modulating membrane cholesterol content. Finally, extraction of cholesterol from the apical plasma membrane of differentiated enterocytes in vitro has a dramatic effect on the structural organization of the plasma membrane [119]. Microvilli are totally absent from enterocytes that were depleted of cholesterol by methyl-β-cyclodextrin. These electron microscope pictures resemble those of ATP8B1-depleted cells which also lack microvilli [36,38,39,41]. CONCLUDING REMARKS

Since the identification of the P4 ATPase family, much progress has been made on the elucidation of their biochemical and  c The Authors Journal compilation  c 2010 Biochemical Society

cellular functions. As illustrated in the present review, the cellular consequences of defects in P4 ATPases vary from vesicle formation deficiencies to membrane structure/stability defects. These defects have secondary consequences, making it difficult to pinpoint the (putative) general biochemical function(s) of the P4 ATPase family, shared by its many members. Recent data have now strongly confirmed that P4 ATPases can operate as phospholipid translocases, when reconstituted in lipid vesicles, possibly in complex with Cdc50 proteins [78,79]. However, it is becoming more and more evident that this activity can be tightly regulated, spatially and temporally. P4 ATPases in multicellular organisms show distinct tissue distribution and specific roles in tissue development and function. In addition, P4 ATPases reside in multiple, often very specific, cellular compartments, from the apical plasma membrane to specialized secretory vesicles. This suggests high spatial regulation of P4 ATPase function. At these specific tissues/cells/organelles, P4 ATPases might have two functions: an ATPase-dependent and a pump-independent function. Furthermore, PS-flippase activity of Drs2p is tightly regulated by a detection system, effectively activating phospholipid translocation at moments and locations where PtdIns4P production, Arf activity and Drs2p coincide. The fact that P4 ATPases are under the control of regulatory proteins and lipids has changed our perspective on temporal and spatial activity of P4 ATPases. Therefore, to understand further the role of other P4 ATPases, it is important not only to study the cellular consequence when they are non-functional, but also to elucidate the specific conditions required for their activation as well as their (subcellular) localization. Finally, studies are needed to define the substrates transported by each P4 ATPase. It is likely that the progress in functional reconstitution of P4 ATPases in liposomes with well-defined composition will aid us in tackling these topics.

FUNDING This study was supported by the Netherlands Organisation for Scientific Research (NWO) [SFJvdG project 016.096.108], the Wilhelmina Children’s Foundation (L.W.J.K.), Utrecht University High Potential Program (L.W.J.K.) and the Dutch Digestive Disease Foundation (L.W.J.K.).

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Received 27 April 2010/13 July 2010; accepted 19 July 2010 Published on the Internet 14 September 2010, doi:10.1042/BJ20100644

 c The Authors Journal compilation  c 2010 Biochemical Society