Differential expression of two ATPases revealed by lipid raft isolation from gills of euryhaline teleosts with different salinity preferences

Highlights

• Lipid raft were extracted from gills of teleosts using nonionic detergents.

• Flotillin-2 expression is differently affected by salinity among teleost species.

• Na⁺, K⁺-ATPase is mainly located in lipid raft fractions enriched in flotillin-2.

• V-type H⁺-ATPase is expressed in both, non-lipid raft and lipid raft fractions.

• Salinity affects V-type H⁺ ATPase expression in non-lipid raft fractions only.

Abstract

In euryhaline teleosts, Na⁺, K⁺-ATPase (NKA) and V-type H + -ATPase A (VHA A) are important ion-transporters located in cell membrane. Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol, sphingolipids, and proteins (e.g., flotillin). Flotillin is a LR-associated protein, commonly used as the LR marker. Previous mammalian studies showed that LR may play a crucial role in ion exchanges. Meanwhile, studies on mammals and rainbow trout showed that NKA were found to be present mainly in LR. However, little is known about LR in fish. Therefore, the present study aimed to investigate the involvement of branchial LR in osmoregulation of tilapia and milkfish, two euryhaline teleosts with different salinity preferences, by (i) extracting LR from the gills of euryhaline teleosts; (ii) detecting the abundance of LR marker protein (flotillin-2) and ion-transporters (NKA and VHA A) in branchial LR and non-LR of fresh water- and seawater-acclimated milkfish and tilapia. The results indicated that the protein abundance of LR marker, flotillin-2, changed with environmental salinities in branchial LR of tilapia. In addition, flotillin-2 and NKA were only found in LR in both tilapia and milkfish gills, while VHA A were mainly present in non-LR. Relative protein abundance of NKA was found to be significantly higher in gills of freshwater milkfish and seawater tilapia, while VHA A was significantly higher in gills of freshwater tilapia and milkfish. This study illustrated differential distribution and salinity-dependent expression of NKA and VHA A in cell membrane of gill tissues of euryhaline teleosts with different salinity preferences.

Introduction

Lipid raft (LR) are membrane microdomains rich in cholesterol and glycoshpingolipids involved in the organization and aggregation of lipid bilayer constituents including transmembrane proteins (Simons and Ikonen, 1997). LR have been reported to be involved in virus entry (Nguyen and Hildreth, 2000; Ono and Freed, 2001), protein trafficking (Brown and London, 1998), ion transport processes (Tillman and Cascio, 2003) and cell signaling (Varma and Mayor, 1998). Because of their high lipid content, i.e., high lipid-to-protein ratio (Brown and Rose, 1992) and relatively low density, the LR fraction can be extracted following non-ionic detergent treatment using density gradients. LR are highly dynamic, usually nanoscale structures that can form bigger raft by means of fusion of small entities (Lingwood and Simons, 2010). Levental et al. (2010) showed that S-acylation, referred to as ‘palmitoylation’ plays an important role in regulating raft affinity. The binding of palmitate with cysteine residues of proteins promotes their insertion into the LR fraction and suggests that palmitoylation plays a critical role in membrane targeting mechanisms involving transmembrane proteins (Resh, 2006). According to Levental et al. (2010) and Contreras et al. (2011), among plasma membrane proteins, about 65% were in the non-raft phase, whereas 12% required palmitoylation for raft phase inclusion, 11% were glycosylphosphatidylinositol (GPI)-anchored in the raft, and the other 11% could be bound to LR lipids such as cholesterol or sphingolipids (SLs).

Lipid compositions of the LR and non-LR fractions are considered to be different as shown in common carp (Cyprinus carpio), i.e., the LR fraction contained a higher percentage of cholesterol and non-polar lipids (Brogden et al., 2014). Non-ionic detergents are thus used to extract LR (also called detergent-resistant membranes (DRM)) from other cell membranes (Eckert et al., 2003; Dalskov et al., 2005; Lingwood et al., 2005). Commonly used detergents for LR extraction in mammalian studies include CHAPS, Tween 20, Triton X-100, Lubrol WX, Brij96, and Brij98, and different detergents that may cause different result of lipid raft isolation due to the dissolvability (Drevot et al., 2002; Madore et al., 1999; Röper et al., 2000; Schuck et al., 2003). Among them, Triton X-100 is the most commonly used detergent for LR extraction (Foster and Chan, 2007; Pike, 2006; Schroeder et al., 1998). Triton X-100 has also been applied in LR research with various protocols in skate (Raja erinacea; Musch et al., 2004), rainbow trout (Lingwood et al., 2005), Atlantic cod (Gadus morhua; Gylfason et al., 2010), goldfish (Carassius auratus; Garcia-Garcia et al., 2012), and common carp (Brogden et al., 2014). Previous studies on fish have not compared different detergents and have not quantified flotillin-2 following LR isolation. Brogden et al. (2014) found that lipid composition of plasma membrane in common carp was different from that in human, and the lipid components were organ-dependent, whether in LR or non-LR regions. In their results, even 0.1% of Triton X-100 cannot perfectly isolate flotillin-2 in LR from non-LR region in all tissue. As a result, it is important to test different concentration of detergents.

Flotillins are LR-associated proteins commonly used as markers for LR. Plasma membrane targeting and clustering of flotillins on LR mainly relies on acylation (myristoylation or palmitoylation) (Banning et al., 2011). Furthermore, they have also been shown to be involved in some basic functions such as trafficking and transport of membrane materials and proteins (Stuermer, 2010). Flotillins are divided into two isoforms. Flotillin-1 (previously named reggie-2) seems to associate with raft by means of the first hydrophobic domain (Liu et al., 2005) and has also been shown to be palmitoylated in Cys34, which is essential for flotillin-1 to locate on the cytoplasmic side of the plasma membrane (Morrow et al., 2002). Flotillin-2 (previously named reggie-1) interacts with the plasma membrane through several sites of palmitoylations and myristoylations, and plays a significant role in the maintenance of membrane raft (Neumann-Giesen et al., 2004; Langhorst et al., 2006). Evidence of flotillin function in fish is scarce. In zebrafish (Danio rerio), von Philipsborn et al. (2005) suggested that flotillin might be involved in development.

The LR plays important roles in ionoregulation and osmoregulation, as shown by proteins responsible for ion transport being localized to LR, including active transport pumps/enzymes (i.e., ATPase) (Tillman and Cascio, 2003; Murtazina et al., 2006). For example, the Na⁺, K⁺-ATPase (NKA) in fish branchial and renal cells (Lingwood et al., 2005; Welker et al., 2007) and vacuolar-type H⁺-ATPase (VHA) in mammalian cells (Lafourcade et al., 2008).

Membrane structure and transmembrane enzyme function are linked because the lipid environment of the enzyme can constrain protein motions required for catalysis, affecting the enzyme catalytic rate (Harris, 1985; Cossins et al., 1986). Among the enzymes/transporters, the NKA catalyzes the transport of Na⁺ and Cl⁻ across epithelia in both absorptive (fresh water; FW) and secretory (seawater; SW) modes in gills of euryhaline teleosts (Marshall, 2002; Perry et al., 2003; Hirose et al., 2003). Changes in branchial NKA activity in euryhaline fish are necessary for acclimation to environmental salinity (Marshall and Bryson, 1998; Kelly et al., 1999; Marshall, 2002; Mancera et al., 2002; Hirose et al., 2003; Lin et al., 2003; Scott et al., 2004). Furthermore, NKA consists of α- and β-subunits (Scheiner-Bobis, 2002). The α-subunit has a molecular weight of about 100 kDa and is considered the catalytic center of the NKA, with binding sites for cations, ATP, and ouabain (NKA inhibitor). The β-subunit, with a molecular weight of 40–60 kDa, can stabilize the structure and regulate the cations affinity of the α-subunit on the plasma membrane (Skou and Esmann, 1992; Abriel et al., 1999). Moreover, reciprocal interactions between NKA and cholesterol or phospholipids have been proposed (Chen et al., 2011; Cornelius, 2008; Cornelius et al., 2015; Haviv et al., 2013; Kravtsova et al., 2015) and NKA distribution on LR has been reported in previous studies on mammalian tissues (Welker et al., 2007). Using the Brij 98 (as a non-ionic detergent) to extract the granulocytes in the brain of rat also revealed the presence of NKA α-subunits in LR (Dalskov et al., 2005). However, there are few studies focused on fish NKA in LR. In gills of rainbow trout (Oncorhynchus mykiss), NKA are expressed in LR when transferred to SW but not detected in LRs in FW individuals, indicating different strategies in ionoregulation between FW- and SW-acclimated rainbow trout (Lingwood et al., 2005). Most reports on NKA α-subunit, however, focused on their localization on the basolateral plasma membrane and their expression when fish encountered different environmental salinities (Lee et al., 2003; Lin et al., 2003). The NKA α-subunit protein abundance in gills of FW-preference Mozambique tilapia (Oreochromis mossambicus) was significantly higher in SW than in FW (Lee et al., 2003), while in gills of SW-preference milkfish (Chanos chanos) it was significantly higher in FW than in SW (Lin et al., 2003).

The VHA is a multi-subunit complex organized into two domains: the 650 kDa cytosolic V₁ domain and the 260 kDa membrane-embedded V₀ domain (Nishi and Forgac, 2002; Forgac, 2007; Toei et al., 2010). In previous mammalian studies, deprivation of cholesterol from LR resulted in the decrease of electrogenic H⁺ efflux by VHA and in synaptic signaling deficiency (Yoshinaka et al., 2004). On frog and insect epithelial cells, VHA contributed to acid-base regulation and osmoregulation (Harvey et al., 1998). In the studies on rainbow trout gills, VHA activity and immunoreactivity decreased when FW trout were acclimated to SW (Lin and Randall, 1993; Lin et al., 1994). In the Atlantic stingray (Dasyatis sabina) and killifish (Fundulus heteroclitus), VHA expressed on the cell membrane was significantly higher in FW than in SW (Piermarini and Evans, 2001; Katoh et al., 2003). As a result, this consistently higher expression of VHA in FW environments is linked to its role in generating an electrical gradient favoring Na⁺ uptake in FW-type ionocytes, additional to its role in acid secretion. In tilapia, bafilomycin, an inhibitor of VHA, has been shown to affect in vivo Na⁺ influx (Fenwick et al., 1999), indicating a key role of this pump in freshwater osmoregulation in tilapia and suggesting an apical location in ionocytes.

To date, most studies on NKA and VHA in fish focus on their relationship with cell membrane, while the relationship between NKA and LR in fish was only reported in the rainbow trout (Lingwood et al., 2005). On the other hands, there is no reference reporting the associations of VHA and LR in fish. According to previous references, this study hypothesized that NKA and VHA, the major ion pumps in fish gills, may be mainly distributed in the LR to be involved in ionoregulation of euryhaline teleosts when acclimated to environments of different salinities. As a result, the present study aims to investigate the presence of LR and their exhibition of NKA and VHA in gills of tilapia (the FW euryhaline teleost) and milkfish (the marine euryhaline teleost), by (i) extracting membrane LR from gills of tilapia and milkfish, with fresh water (FW) and seawater (SW) preferences, respectively, and (ii) assessing differential NKA and VHA expression in branchial LR between FW- and SW-acclimated milkfish and tilapia. This study provides the evidence for expression of critical ion transporters in LR of euryhaline teleosts with different salinity preferences. Differential expression of flotillin-2 (the LR marker) and two ATPases, the ATP-consuming NKA and VHA, in gills of euryhaline fish will further clarify the ionoregulation roles of branchial LR in environments of different salinities.