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

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.