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Am J Physiol Regul Integr Comp Physiol 294: R1657-R1665, 2008. First published March 12, 2008; doi:10.1152/ajpregu.00837.2007
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Volume expansion stimulates monoubiquitination and endocytosis of surface-expressed skate anion-exchanger isoform

Mark W. Musch,1,2 Amanda B. Puffer,1,3 and Leon Goldstein1,3

1Mount Desert Island Biological Laboratory, Salsbury Cove, Maine; 2Inflammatory Bowel Disease Research Center, Martin Boyer Laboratories, Department of Medicine, The University of Chicago, Chicago, Illinois; and 3Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island

Submitted 20 November 2007 ; accepted in final form 10 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In hyposmotic medium, skate erythrocytes swell and then lose taurine and other solutes with obligate water to achieve a regulatory volume decrease (RVD) over a 90-min period. The skate erythrocyte anion-exchanger isoform 1 (skAE1) participates in the RVD, and increased surface expression after hyposmolality-induced volume expansion occurs within 5 min but decreases to baseline within 120 min. The subsequent fate of skAE1 is the focus of these studies. SkAE1 sent to the surface becomes monoubiquitinated, a modification that is present while skAE1 is associated with clathrin and Rab5 but is removed before skAE1 is passed to the Rab4 compartment. Endocytosis of skAE1 involves clathrin-mediated internalization. Surface plasma membrane skAE1 forms tetramers and demonstrates increased tyrosine phosphorylation, and both of these processes decrease before skAE1 appears in the Rab5 compartment. Volume expansion-stimulated surface skAE1 comes from an intracellular pool in a buoyant membrane fraction resistant to nonionic detergent extraction (DRM), and the amount of skAE1 increases in this buoyant DRM fraction on the surface. Clathrin heavy chain is found largely in the erythrocyte DRM, but in dense, rather than buoyant, fractions. Rab5- and Rab4-containing membranes are largely detergent soluble, suggesting that as skAE1 is passed to clathrin and then to Rab5 compartments, the membrane microdomain composition changes. The present studies demonstrate that skAE1, which appears on the surface after hyposmolality-induced volume expansion, is monoubiquitinated, a modification that may serve as a signal for removal of skAE1 from the surface. This modification is eliminated after clathrin-mediated removal of skAE1 in a membrane domain containing Rab5, potentially permitting recycling and reuse of skAE1.

regulatory volume decrease; clathrin; Rab5; Rab4; tetramerization; tyrosine phosphorylation; membrane domains

THE SKATE ERYTHROCYTE has been used as a model of cellular regulation of transporters involved in a process termed the regulatory volume decrease (RVD), whereby cells that are swollen due to exposure to a hyposmotic medium lose solutes and obligate water. The permeability pathways used to accomplish solute loss for the RVD may differ with cell type but include KCl cotransporters, K+ channels, and various other channels that allow passage of a number of organic osmolytes. In fact, identification of the volume-stimulated osmolyte anion channel(s) has been controversial, and potential identities include a number of Cl– channels, a protein designated pICln, and certain anion exchangers (30, 36, 37). In the skate erythrocyte, which possesses three related isoforms of the anion-exchanger family (skAE1, skAE2, and skAE3) (13), it appears that at least one isoform of the anion exchanger, skAE1, which is the most homologous to the human erythrocyte anion exchanger, can participate or allow permeation of small molecules such as taurine, betaine, sorbitol, and others. Indeed, skAE1 expressed in Xenopus laevis oocytes confers a volume-regulated permeability to taurine that resembles the permeability and regulation of the native skate erythrocyte volume-stimulated taurine permeability pathway (19, 20).

Under basal conditions, some anion exchange is required by the skate erythrocyte. However, after volume expansion, additional anion exchange is required, and the need for surface plasma membrane-expressed skAE1 increases. Volume expansion-stimulated taurine efflux returns to near-basal levels within 60–90 min, and cellular volume returns to near-basal levels within 120 min (8). Therefore, the need for surface-expressed skAE1 is transient.

The process of endocytosis has been widely studied in proteins with diverse function on the surface membrane, including receptors for growth factors, neurotransmitters, and certain G protein-coupled receptors (10, 42, 48), as well as receptors for a number of transport proteins, including CFTR (1, 11, 31, 39–41), ROMK channels (22), the epithelial Na+ channel (ENaC) (35, 44), GLUT4 (3, 4, 12), and also a Saccharomyces monosaccharide transporter (17, 23). A number of pathways may mediate surface protein removal. These include pathways that involve clathrin or DRM domains and, because of high cholesterol content, are less dense than other regions of the plasma membrane and, therefore, sometimes termed buoyant rafts (5, 15, 24). Proteins that have been removed from the surface membrane may be directed to lysosomal or proteosomal degradation, or they may be recycled. The particular fate of an endocytosed protein may be regulated through interaction with a large variety of proteins. A large number of proteins that may be involved have been identified, including early endosomal antigen, Rme-1, and the highly related small GTP-binding proteins of the Rab family. Although incompletely understood, the fate of an endocytosed protein may be regulated through protein-protein interaction, encouraging routing through specific pathways. This directional movement can be regulated by the addition of a modifier, e.g., ubiquitin groups, to the endocytosed protein (7, 32, 43). Additionally, interaction can be regulated through phosphorylation and dephosphorylation of specific amino acid residues of the endocytosed protein, as well as adaptor proteins involved in endocytosis (9, 21).

The goal of the present studies was to determine the fate of skAE1 at various times after volume expansion induced by hyposmolality. The present studies demonstrate that as the erythrocyte achieves taurine efflux and the RVD, skAE1 is removed from the surface by a clathrin-dependent pathway. At later times, skAE1 is found in membranes containing the early endosomal marker Rab5 and, later, in membranes containing Rab4, suggesting that skAE1 may be recycled. Monoubiquitination of skAE1 on the membrane surface may be an important signal for skAE1 endocytosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and treatment of skate erythrocytes. Little skate (Raja erinacea) were caught in the Gulf of Maine and obtained from the Canadian Fisheries Laboratory (St. Andrews, NB, Canada). All procedures were performed in accordance with protocols approved by our Institutional Animal Care and Use Committee. Skates were kept in 15°C running seawater until use at Mt. Desert Island Biological Laboratory. Blood was removed from a tail vessel into a heparinized syringe. Cells were pelleted twice (400 g for 2 min at room temperature), and the plasma and buffy coat were removed by aspiration. Erythrocytes were resuspended at 50% hematocrit in isosmotic elasmobranch incubation medium [940 EIM; in mmol/l: 300 NaCl, 5.22 KCl, 2.7 MgSO4, 5 CaCl2, 370 urea, and 15 Tris (pH 7.4)]. For incubations, cells were diluted to 10% hematocrit with 940 EIM or hyposmotic elasmobranch incubation medium (460 EIM; NaCl was reduced to 100 mmol/l and urea to 250 mmol/l). One-milliliter samples (100 µl of packed cells) were removed at various times for analysis of skAE1 association with various membrane vesicle populations or rapidly chilled for surface biotinylation or isolation of buoyant membrane domains.

Taurine efflux studies. Efflux studies were used to determine the time course of hyposmolality-stimulated taurine transport. For efflux studies, cells were loaded for 2 h in 940 EIM to which 1 µCi/ml [3H]taurine (Perkin Elmer Life Sciences, Boston, MA) was added at 15°C. For removal of extracellular isotope, cells were washed in 940 EIM with 10 mM taurine, then in 940 EIM with 1 mM taurine, and, finally, in 940 EIM with 0.1 mM taurine. Cells were resuspended at 50% hematocrit in 940 EIM and then diluted to 10% hematocrit in 940 or 460 EIM. Aliquots of cells were removed at various times and immediately pelleted at 14,000 g at room temperature for 20 s. The supernatant was removed as taurine was released, and the cell pellet was weighed and extracted with 0.7% (wt/vol) perchloric acid for extraction of cellular isotope. Proteins were allowed to precipitate in the acid on ice for 30 min and removed by centrifugation (14,000 g for 2 min at room temperature); then the supernatant was removed and counted for determination of cellular isotope content. Effluxes were calculated as previously described (15).

Membrane compartment pull-down studies. Membrane pull-down studies were performed to determine the association of skAE1 with membrane compartments/domains that contain clathrin or Rab proteins. Skate erythrocytes were swollen in hyposmotic medium, and aliquots were rapidly pelleted (14,000 g for 20 s) at various times. Some cells were kept in 940 EIM and used as controls. EIM was aspirated, and cell pellets were frozen in a dry ice-ethanol bath. For isolation of ghosts, samples in buffer were thawed on ice [10 mM Tris (pH 7.4) and 5 mM EDTA, with 1 mM PMSF, 5 mM NaF, 10 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, and complete protease inhibitor (Roche Molecular Biochemicals)]. Ghosts were made by pelleting (10,000 g for 15 s) and resuspension in 10 ml of buffer for three cycles and repelleting (until no hemoglobin was present in the pellet). Ghosts were sonicated by two 20-s pulses with a Branson sonifier microprobe at a setting of 5. Mitochondria and nuclei were removed by centrifugation (10,000 g for 30 s at 4°C), and microsomal membranes were isolated by centrifugation (100,000 g for 20 min at 4°C). Membranes were resuspended in 500 µl of immunoprecipitation buffer. Nonspecific binding was cleared by rotation of 50 µl of 50% (wt/vol) slurry of Pansorbin Staphylococcus aureus bacteria bearing protein A with the sample for 30 min at 4°C. Pansorbin cells were pelleted (400 g for 30 s at 4°C), and the supernatant was removed and added to Sepharose beads to which various antibodies had been coupled using the Seize X kit (Pierce Chemical, Rockford, IL) and rotated overnight at 4°C. Anti-Rab4 (Stressgen, Victoria, BC, Canada) and anti-Rab5 and anti-heavy chain clathrin (BD Transduction Labs, Lexington, KY) antibodies were chosen, inasmuch as they recognize proteins of appropriate molecular masses in skate erythrocytes (compared with mammalian cells). The Rab4 antibody is a rabbit polyclonal antiserum directed against full-length human Rab4, the Rab5 polyclonal antiserum was raised against a peptide (amino acids 182-197) of rat Rab5, and the clathrin heavy chain monoclonal antibody was raised against a segment (amino acids 4-171) of the human heavy chain of clathrin. Both Rab antibodies recognized predominantly a ~25-kDa protein, and the anti-heavy chain of clathrin antibody recognized nearly exclusively a ~185-kDa protein. Beads and associated proteins were pelleted and washed four times with buffer, and samples were eluted by heating to 65°C with 50 µl of 1x Laemmli stop solution. Samples were resolved on SDS-polyacrylamide gels of various percentages, and Western blots were immediately generated by transfer of proteins to polyvinylidene difluoride membranes in 1x Towbin's buffer. Blots were blocked in 3% (wt/vol) bovine serum albumin in Tween containing Tris-buffered saline [TTBS, 10 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, and 5 mmol/l KCl with 0.2% (vol/vol) Tween 20] and incubated overnight in TBS with various antibodies, including those listed above for immunoprecipitation, as well as anti-skAE1 antiserum (directed to the last carboxy-terminal 17 amino acids) (7) or anti-phosphotyrosine murine monoclonal antibody 4G10 (Upstate Biotechnology). Blots were washed four times for 10 min each in TTBS and then for 60 min at room temperature with species-appropriate peroxidase-conjugated secondary antibodies, washed three times in TTBS and once in TBS without Tween, and developed using an enhanced chemiluminescence system (Supersignal, Pierce Chemical). Blots were washed twice in Restore Western blot stripping buffer (Pierce Chemical) and analyzed with a second primary antibody exactly as described above.

Surface expression and biotinylation. The cell-impermeant agent sulfo-NHS-biotin (Pierce Chemical) was used to label proteins that may be exposed to the exterior surface. Cells were removed and immediately chilled on ice and pelleted (2,000 g for 2 min). The supernatant was aspirated and exchanged for 940 or 460 EIM, where HEPES replaced Tris and mannitol replaced urea. Since biotin reacts with amines, this buffer switch was required. Reactions were kept for 30 min at 4°C to minimize endocytosis and exocytosis. Reactions were stopped by addition of 10 µl of 1 M Tris (pH 7.4). For isolation of biotinylated material, microsomal membranes were prepared as described above and solubilized with 500 µl of buffer [150 mmol/l NaCl, 2 mmol/l EDTA, 10 mmol/l HEPES (pH 7.4), 1 mmol/l phenylmethylsulfonyl fluoride, 10 mmol/l β-glycerophosphate, 5 mmol/l NaF, 1% (vol/vol) Triton X-100, and complete protease inhibitor cocktail]. Proteins were mixed with 30 µl of streptavidin-agarose and rotated at 4°C for 120 min, and the beads were pelleted (14,000 g for 20 s) and washed three times in buffer. Samples were eluted by heating in 1x Laemmli stop solution, proteins were resolved on SDS-PAGE, and Western blots were used for analysis of skAE1.

Tetramer formation studies. The homobifunctional cross-linking agent BS3 (Pierce Chemical) was used to determine whether skAE1 in various fractions had formed tetramers. Microsomal membranes were isolated as described above and resuspended in buffer [10 mM HEPES (pH 7.4) and 2 mM EDTA, with phosphatase and protease inhibitors] as described above. BS3 was dissolved in a minimal amount of dimethylsulfoxide and added to a final concentration of 5 mM. Reactions were allowed to proceed for 30 min and stopped by addition of 10 µl of 1 M Tris (pH 7.4). When appropriate, clathrin, Rab5, or Rab4 antibody was used to isolate fractions. Samples were resolved on 5% SDS-polyacrylamide gels and transferred, and Western blots were analyzed with the skAE1 antiserum.

Lipid raft isolation. Regions of membranes with low density (due to high cholesterol content, which caused tight packing of phospho- and sphingolipids) were isolated on 5–30% (wt/vol) continuous sucrose density gradients as described previously (29). Microsomal membranes from cells that had been surface biotinylated were isolated and resuspended in gradient buffer [10 mM Tris (pH 7.4) and 2 mM EDTA] with 40% (wt/vol) sucrose. A continuous gradient of 5–30% (wt/vol) sucrose in gradient buffer was layered on top, and gradients were spun at 37,000 g at 4°C for 18 h. Fractions (0.5 ml) were removed, and streptavidin-agarose (25 µl) was added to half of the fractions, which were rotated at 4°C for 120 min, pelleted (14,000 g for 20 s), and washed four times, and biotinylated material was eluted by heating with Laemmli stop solution for analysis of surface-expressed skAE1. The other half of the fractions was concentrated to 20% of volume, mixed with Laemmli stop solution, and heated to 65°C for 10 min for analysis of total skAE1. Total and surface-expressed skAE1 was analyzed by Western blotting as described above. For analysis of skAE1 ubiquitination, blots were stripped using Restore Western blot solution and analyzed using murine monoclonal P4D1 or FK2 anti-ubiquitin antibodies.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SkAE1 appears rapidly on the surface, but surface expression decreases along with hyposmolality-stimulated taurine efflux. As shown in Fig. 1A, exposure of skate erythrocytes to hyposmotic medium (460 EIM) rapidly stimulates taurine efflux. The rate of taurine efflux is greatest over the first 15–30 min, then decreases, and is nearly complete by 90 min. Figure 1A also shows the percentage of taurine remaining in the cells at each point after exposure to hyposmotic medium. The erythrocytes lose a significant amount of taurine; however, a large gradient for taurine to exit the cell remains, even after 240 min. Therefore, a greatly diminished gradient does not explain the decrease in taurine efflux. Rather, a change in the permeability path for taurine likely explains the transience of the efflux. Since skAE1 mediates the hyposmolality-stimulated taurine efflux, the surface expression of this transporter was determined. The abundance of skAE1 on the surface increases within 5 min, increases up to 30 min, and returns to near baseline by 90 min (Fig. 1B), similar to the kinetics of volume-stimulated taurine efflux.


Figure 1
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  		Fig. 1. Time course of taurine efflux and surface skate anion-exchanger isoform 1 (skAE1) expression after hyposmolality-induced volume expansion. A: skate erythrocytes (RBCs) were loaded with [3H]taurine and then kept in isosmotic medium (940 EIM) or placed in hyposmotic medium (460 EIM), and [3H]taurine released from the cells (efflux) or taurine remaining in the cells was measured at various times by centrifugation. Values are means ± SE for 4 separate experiments. B: skate erythrocytes were kept under isosmotic conditions (940 EIM) or volume expanded with half-osmolar medium (460 EIM) for various times. Cells were chilled, rapidly pelleted, and resuspended in osmolarity-appropriate EIM with Tris replaced by HEPES and urea replaced by mannitol, and the cell-impermeant sulfo-NHS-biotin (1 mM) used to biotinylate surface proteins. After 30 min of biotinylation, cells were pelleted, ghosts were prepared, and biotinylated proteins were isolated from microsomal membranes with use of streptavidin-agarose. SkAE1 was analyzed in Western blots from biotinylated proteins. Image is representative of results from 4 separate experiments. 120C represents untreated 240 min isosmotic control. Values are means ± SE. Isosmolality at time 0 (Iso t0) was set to 100. *P < 0.05; +P < 0.01; ++P < 0.001 vs. time 0 isosmotic (by ANOVA).

 
SkAE1 is internalized via clathrin, Rab5, and Rab4 pathways for recycling. To determine whether clathrin-mediated endocytosis is involved in retrieval of surface skAE1, membranes containing the heavy chain of clathrin were isolated. In isosmotic medium, skAE1 is present, but in very low abundance, in the clathrin-containing membrane compartment; however, skAE1 abundance increases within 15 min of volume expansion and transiently increases and then decreases in the clathrin-containing compartment (Fig. 2A ). The abundance of skAE1 increases at later times in a membrane compartment containing Rab5, an early endosomal marker (Fig. 2B). From Rab5, endocytosed proteins may be directed to many pathways, including recycling via a Rab4-dependent pathway. The abundance of skAE1 in Rab4-containing membranes increases after volume expansion upon maximal skAE1 abundance in the Rab5 compartment (Fig. 2C). All membrane pull-down studies confirmed equal clathrin, Rab5, and Rab4 at all time points in their respective analyses.


Figure 2
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  		Fig. 2. Time course of skAE1 appearance in membrane compartments containing heavy chain (HC) of clathrin (A), Rab5 (B), or Rab4 (C). Skate erythrocytes were volume expanded in hyposmotic (460) EIM or kept in isosmotic conditions (940 EIM). An aliquot of cells was removed at various times and rapidly pelleted, and the cell pellet was frozen to be thawed later to make microsomal membranes and pull-down membranes containing the heavy chain of clathrin, Rab5, or Rab4. Pulled-down material was eluted, and Western blots of the proteins were generated and probed for skAE1, as well as for the protein pulled down by the antibody. Images are representative of 3 separate experiments. IP, immunoprecipitation. 240C represents untreated 240 min isosmotic control. Values are means ± SE. *P < 0.05, +P < 0.01; ++P < 0.001 vs. time 0 isosmotic (by ANOVA).

 
Monoubiquitination of skAE1 occurs at the surface but not in the Rab5-containing compartment. For detection of ubiquitination, two different murine monoclonal antibodies were used. The antibody P4D1 recognizes mono- and polyubiquitination, whereas FK2 recognizes only polyubiquitination (14). Under isosmotic and hyposmotic conditions, the level of protein ubiquitination is very low, but ubiquitination can be increased under both conditions by incubation with the proteasome inhibitor MG-132 (30 µM) for 30 min (Fig. 3A). Ubiquitination of certain proteins was increased by hyposmotic exposure, most apparently without proteasome inhibition by the P4D1 antibody (Fig. 3A).


Figure 3
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  		Fig. 3. SkAE1 monoubiquitination increases with hyposmotic volume expansion and is not removed until skAE1 passes from the Rab5- to Rab4-containing compartment. Erythrocytes were volume expanded in 460 EIM (Hypo) or left in 940 EIM (Iso). A: skAE1 ubiquitination in microsomal membranes was analyzed with and without pretreatment with the proteasome inhibitor MG-132 (30 µM) for 30 min, and blots were probed with anti-ubiquitin monoclonal antibodies P4D1 and FK2. B: skAE1 was immunoprecipitated from total membranes from untreated cells or erythrocytes labeled with biotin for 30 min and then reacted with streptavidin-agarose for isolation of surface skAE1. Western blots were generated from these immunoprecipitates and analyzed for skAE1 and then for ubiquitination using P4D1 antibody. C: SkAE1 was immunoprecipitated from clathrin, Rab5, and Rab4 pull-downs and analyzed by Western blots first for skAE1 and then for ubiquitination using P4D1 antibody. Images are representative of 3 separate experiments. Densitometric values were set to 100 for the isosmotic condition for each group, and the hyposmotic value was calculated as an increase. Values are means ± SE. ++P < 0.001 vs. time 0 isosmotic and hyposmotic (by ANOVA).

 
In cells without proteosome inhibition upon volume expansion, ubiquitination was further investigated by immunoprecipitation of skAE1 (Fig. 3B). In addition, use of cells that had been surface biotinylated allowed analysis of surface skAE1. Ubiquitination of skAE1 increased dramatically upon hyposmotic volume expansion, and surface skAE1 also demonstrated a large increase in ubiquitination (Fig. 3B). This ubiquitination appears to be monoubiquitination, inasmuch as a strong reaction was observed with the P4D1 antibody, but little or no reaction was observed with the FK2 antibody (Fig. 3B). To determine which membrane compartments contained monoubiquitinated skAE1, we analyzed membrane pull-downs of clathrin, Rab5, and Rab4 at 15, 30, and 60 min, respectively, after hyposmolality-induced volume expansion, as well as an isosmotic control at the same time points (time points were chosen as maximal abundance of skAE1 after volume expansion). SkAE1 was monoubiquitinated while it was in the clathrin-containing complex, as well as the Rab5-containing compartment, but not by the time it reached the Rab4-containing compartment (Fig. 3C), suggesting that a ubiquitinase is active in the Rab5-containing membrane compartment.

SkAE1 tetramers do not appear in either Rab compartment. Formation of a tetramer appears to be pivotal for skAE1 mediation of taurine efflux (27, 28). To determine at which step tetramers are dismantled during endocytosis, skAE1 was cross-linked with BS3 and analyzed in microsomal membranes, as well as clathrin-, Rab5-, and Rab4-containing membrane pull-downs. SkAE1 was analyzed in clathrin, Rab5, and Rab4 compartments at 15, 30, and 60 min, respectively, which are times of maximal skAE1 abundance in these compartments. As previously demonstrated, hyposmotic conditions stimulated the formation of tetramers (analyzed in total microsomal membranes 15 min after volume expansion; Fig. 4). SkAE1 tetramers were also detected in the clathrin pull-down but could not be detected in the Rab5 or Rab4 pull-down (Fig. 4).


Figure 4
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  		Fig. 4. SkAE1 tetramer is present when skAE1 is in clathrin-containing membranes, but not when skAE1 reaches Rab5-containing membranes. Erythrocytes were volume expanded in 460 EIM or left in 940 EIM. Ghosts and microsomal membranes were made from cells after 15 min for total or clathrin-associated membrane compartment, after 30 min for Rab5-containing compartment, or after 60 min for Rab4-containing compartment. Microsomal membranes were reacted with the bifunctional cross-linker BS3 for 30 min. Membrane compartments were isolated by pull-downs with specific antibodies. Images are representative of 3 separate experiments.

 
Hyposmolality-stimulated skAE1 tyrosine phosphosphorylation decreases before the clathrin-containing compartment. SkAE1 possesses many tyrosine residues, some of which are phosphorylated under basal conditions; however, tyrosine phosphorylation increases upon cell volume expansion (28). To determine at which step tyrosine dephosphorylation occurs, we analyzed skAE1 from the membrane pull-downs for clathrin, Rab5, and Rab4, as total microsomal membranes. Analysis of skAE1 from total microsomal membranes or surface-expressed skAE1 shows an increase in skAE1 tyrosine phosphorylation in hyposmotic medium (Fig. 5 ). SkAE1 in the clathrin, but not the Rab5 or Rab4, pull-down demonstrated increased tyrosine phosphorylation compared with skAE1 abundance (Fig. 5), suggesting that a tyrosine phosphatase may act on skAE1 before the Rab5 compartment.


Figure 5
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  		Fig. 5. Increased tyrosine phosphorylation is present when skAE1 is in clathrin-containing membranes, but not when skAE1 reaches Rab5-containing membranes. Erythrocytes were volume expanded in 460 EIM (H) or left in 940 EIM (I). Ghosts and microsomal membranes were made from cells after 15 min for total or clathrin-associated membrane compartment, after 30 min for Rab5-containing compartment, or after 60 min for Rab4-containing compartment; membrane compartments were isolated by pull-downs with specific antibodies. Western blots of pulled-down proteins were analyzed for skAE1 and then for phosphotyrosine ({alpha}-PY). Images are representative of 3 separate experiments. Densitometric values were set to 100 for the isosmotic condition for each group, and hyposmotic value was calculated as an increase. Values are means ± SE. *P < 0.05; ++P < 0.001, isosmotic vs. hyposmotic (by ANOVA).

 
SkAE1 passes between multiple membrane microdomains. To further understand the events that occur during skAE1 endocytosis, we investigated the nature of the membrane regions containing clathrin, Rab5, and Rab4, inasmuch as these are regions "traversed" by skAE1 during endocytosis. A majority of the heavy chain of clathrin was present in high-density membrane material that is insoluble (resistant) to nonionic detergent, whereas a majority of Rab5 and Rab4 was in the detergent-soluble membrane fractions (Fig. 6). Therefore, the membrane domains containing the associated accessory proteins required for endocytosis are different, and skAE1 must pass from buoyant- to dense-surface DRM (while associated with clathrin) and then to detergent-soluble intracellular membranes (while associated with Rab5 and Rab4). Eventually, the pool of skAE1 could pass back to the pool used for exocytosis, which is intracellular but is a buoyant DRM fraction.


Figure 6
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  		Fig. 6. Detergent solubilities of proteins involved in skAE1 endocytosis. Membranes were solubilized using 1% (vol/vol) Triton X-100 on ice for 30 min, and equal amounts of protein were analyzed on Western blots for heavy chain of clathrin and Rab5 and Rab4. Detergent-resistant fraction was additionally separated by density on a 5–30% sucrose gradient, and fractions were removed and analyzed by Western blotting for heavy chain of clathrin. Images are representative of 3 separate isolations. Densitometric values were added, and percentage in detergent-soluble (S) and -insoluble (I) fractions was calculated. Values are means ± SE. +P < 0.01; ++P < 0.001, S vs. I (by paired Student's t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Under isosmotic conditions, the need for the anion-exchange function of the skAE1 is served by skAE1 expression on the cell surface, which is ~20% of total cell skAE1 expression. Upon volume expansion stimulated by hyposmotic medium, skAE1 is "recruited" to the surface from an intracellular reservoir. The present studies demonstrate that surface skAE1 is removed from the surface, inasmuch as the need for volume-expanded stimulation of taurine flux has resulted in the RVD. An important signal in skAE1 appears to be monoubiquitination, as has been demonstrated for a small number of surface-expressed proteins of diverse functions (14, 16, 17, 42). Poly- and monoubiquitination of many different proteins has been investigated. Many proteins, including a variety of surface membrane proteins, are polyubiquitinated, and these polyubiquitinated proteins may signal processes such as proteosomal or lysosomal degradation and may also assist in recycling of certain proteins. Monoubiquitination has been observed in a more limited number of studies, but, as cited above, increasing numbers of surface membrane proteins may be monoubiquitinated, and the fates of these proteins vary. In the present studies, surface monoubiquitinated skAE1 may be found in many membrane domains, including the Triton-insoluble material. In the DRM, monoubiquitinated skAE1 is found in buoyant and dense fractions. The dense fraction also contains clathrin, an important pathway for retrieval of many proteins from the surface plasma membrane. SkAE1 appears in a clathrin-containing compartment within 15 min after volume expansion in hyposmotic medium, demonstrating that at least one pathway for endocytosis involves clathrin. Subsequently, skAE1 appears in a Rab5-containing membrane compartment and, later, in a Rab4-containing compartment. Monoubiquitinated skAE1 is present on the surface and is also retained in the clathrin (which is initially on the surface), as well as the Rab5, compartment but is removed before skAE1 reaches the Rab4 compartment (both Rab compartments are intracellular). It is possible that elimination of monoubiquitination is important for skAE1 recycling. Mono- and polyubiquitination may be a signal for lysosomal and proteosomal protein degradation (2, 14, 32), and deubiquitination may be important for prevention of skAE1 degradation.

Endocytosis of skAE1 is mediated by clathrin, but it cannot be assumed that this is the only pathway for endocytosis. A number of non-clathrin-dependent pathways have been identified, and some of these involve endocytosis directly from surface membrane lipid raft regions. In addition, other proteins are likely to be involved in the endocytosis of skAE1. In cases of clathrin-dependent and -independent endocytosis, a member of the epsin family of proteins may be involved (6, 38). Epsin-dependent endocytosis has been demonstrated in transport proteins, including the ENaC (35, 44) and the dopamine transporter (34). Epsin proteins have ubiquitin interaction motifs and may be ubiquitinated (2, 32, 43); therefore, they might have a role as accessory proteins in monoubiquitination of skAE1 before endocytosis. However, whether epsin proteins are able to bind mono- vs. polyubiquitinated proteins remains controversial. We have tested a number of epsin antibodies in skate membranes and have been unable to detect epsin, suggesting that the skate homolog may be sufficiently different that it is not recognized by antibodies to mammalian epsin. Also relevant to potential differences in skate proteins is the failure of anti-Rab11 antibody to detect a skate erythrocyte protein. In some cases, proteins to be recycled, including certain transport proteins, e.g., CFTR, are passed through a Rab11-containing complex (11, 41). The use of a cell that cannot be cultured from a species where only a small number of proteins have been cloned presents experimental challenges. First, as the present studies demonstrate, antibodies raised to mammalian proteins may not recognize the phylogenetically older form in elasmobranches. Likely homologous proteins are expressed; however, they have not been cloned. Since the proteins have not been cloned, use of technologies to silence expression is not possible. This leads to the second consideration: dominant-negative constructs cannot be developed, and RNA silencing cannot be performed. These technologies require that cells survive ≥24 h in vitro. The ability of the skate erythrocyte to manifest RVD after 8 h decreases dramatically, even when kept as drawn blood. Cells kept in medium with many substrates, even substrate including 10% skate serum, do not respond well to hyposmotic medium after 8 h. Heterologous expression systems using cultured cells, such as HEK cells and oocytes, may provide a model, but necessary accessory proteins, potentially unique to the skate erythrocyte, will also need to be cloned and, possibly, coexpressed.

The present studies help elucidate the events that allow the skate erythrocyte to manifest RVD. A model for alterations of skAE1 is presented in Fig. 7. Under unstimulated conditions, some skAE1 resides on the surface, as would be needed for anion exchange; however, the majority of skAE1 is intracellular. Under these unstimulated conditions, skAE1 binds the cytoskeletal protein band 4.1 with high affinity. It is unknown whether surface or intracellular skAE1 binds 4.1, but we hypothesize that band 4.1 keeps skAE1 in an intracellular reservoir. The band 4.1-skAE1 interaction is disrupted rapidly upon volume expansion, and skAE1 forms high-affinity interactions with ankyrin, a different cytoskeletal protein. At the same time, the level of skAE1 tyrosine phosphorylation increases. The tyrosine kinase p72syk appears to be involved, since piceatannol, a relatively specific inhibitor of p72syk, blocks the volume-expanded taurine efflux and hyposmolality-stimulated increase in skAE1 tyrosine phosphorylation. Nearly simultaneously with increased tyrosine phosphorylation, skAE1 forms tetramers. Under unstimulated conditions, skAE1 forms dimers (~50% of the total skAE1); however, upon volume expansion, skAE1 tetramers form. SkAE1 tetramer formation parallels the time course of taurine efflux. In addition, tetramer formation depends on increased skAE1 tyrosine phosphorylation.


Figure 7
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  		Fig. 7. Model of volume-expanded stimulation of skAE1 exocytosis and endocytosis. DRM, nonionic detergent-resistant membrane.

 
The present study demonstrates that tetramers are stable until the skAE1 is internalized. The tetramers are present while skAE1 associates with clathrin-containing membranes but are dismantled by the time skAE1 reaches the intracellular Rab5-containing membrane compartment. The level of tyrosine phosphorylation is also high while skAE1 is in the clathrin-containing compartment but decreases when skAE1 is passed to the Rab5 compartment. The nature of the tyrosine phosphatase is unknown. Monoubiquitination is a signal that appears to direct skAE1 to the clathrin-containing compartment and subsequent endocytosis. Monoubiquitination is present until skAE1 reaches the Rab5-containing compartment. We hypothesize that deubiquitination could be important in passage of skAE1 to the Rab4-containing compartment. It is believed that Rab4 directs proteins to be recycled. It would be advantageous for the skate erythrocyte to recycle skAE1, inasmuch as skate erythrocytes have limited transcriptional (13) or translational activity, as evidenced by the small amount of [35S]methionine incorporated by skate erythrocytes (26). Skate erythrocytes have a life of many months in the bloodstream, and the ability to reuse the exchanger could be important for future function.

Erythrocytes from the little skate R. erinacea represent one model of RVD, but the importance and regulation of skAE1 are likely not limited to skates. There is significant controversy concerning the identification of transport proteins that regulate osmolyte loss upon volume expansion. A number of Cl– channels have been proposed to mediate osmolyte loss in a variety of cell systems (30, 36, 37). It should not be assumed that only one transporter might mediate osmolyte loss upon volume expansion. Erythrocytes are distinct in the transport proteins they express and are the only cells to express "band 3," the anion exchanger that is the most abundant of mammalian membrane erythrocyte proteins. The skate erythrocyte is unusual, in that three anion-exchanger isoforms are expressed: skAE1, which is most homologous to human, mouse, and trout AE1; skAE2, which is more homologous to avian erythrocyte AE1; and skAE3. We have investigated skAE1 to the greatest extent and have demonstrated that skAE1 can mediate volume-expanded taurine efflux. Future investigations will determine whether the other anion-exchanger isoforms may mediate taurine efflux or play a role in skAE1 regulation. Additionally, those domains of skAE1 that are pivotal for formation of a taurine permeability pathway will also be elucidated.

Perspectives and Significance

The present studies are important, inasmuch as they help establish monoubiquitination as a potential signal for endocytosis of surface transport proteins. The process of ubiquitination is phylogenetically conserved and has been used as a signal for protein trafficking since prokaryotes. Mono- and polyubiquitination may have different roles, and it is possible that polyubiquitin modifications may direct a protein to degradation by the proteasome or lysosome, whereas monoubiquitin in some cases may allow protein recycling. SkAE1 may represent a protein that may serve many functions, as well as a protein that should be recycled by a cell with limited translational activity but a long life.


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This work was supported by National Science Foundation Grant IBN-0412702 (to L. Goldstein) and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38510 and DK-47722 (to M. W. Musch).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Goldstein, Box G-B311, Dept. of Molecular Pharmacology, Physiology, and Biotechnology, Brown Univ., Providence, RI 02912 (e-mail: Leon_Goldstein{at}Brown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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