One response to hypertonic stress in the renal medulla and MDCK cells is the upregulation of betaine transporter (BGT1) synthesis, followed by trafficking to the plasma membrane (PM) and an increase in betaine transport. Upregulation of BGT1 was enhanced by inhibitors of phosphatases PP1 and PP2A and was attenuated by inhibitors of protein kinase C, suggesting an important role for phosphorylation reactions. This was tested using mutants of BGT1 tagged with EGFP. The PM trafficking motifs of BGT1 reside near the C terminus, and truncation at lysine560 resulted in a protein that remained intracellular during hypertonic stress. This K560 mutant colocalized with endoplasmic reticulum (ER). Substitution of alanine at Thr40, a putative phosphorylation site, also prevented trafficking to the PM during hypertonic stress. Live-cell imaging showed that T40A was not retained in the ER and colocalized with markers for Golgi and endosomes. In contrast, substitution of aspartate or glutamate at Thr40, to mimic phosphorylation, restored normal trafficking to the PM. HEK293 cells transfected with K560 or T40A mutants had 10% of the GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Normal BGT1 trafficking likely requires phosphorylation at Thr40 in addition to C-terminal motifs. 1. Introduction The hypertonicity of interstitial fluid in the renal medulla, more than 1,000？mosmol/kg in humans, is essential for excretion of concentrated urine and for conserving water for the body. Mammalian cells in a hypertonic environment will lose water rapidly and shrink which, if prolonged, leads to misfolding and aggregation of proteins, cell cycle delay, apoptosis, and necrosis [1–3]. One cellular defense against hypertonicity is intracellular accumulation of organic compounds termed osmolytes which do not interfere with normal cell functions. As the intracellular osmolyte concentrations increase, the cell volume also increases due to osmotic entry of water, and the intracellular ionic strength is reduced. The process is slow, up to 24？h, because gene expression is involved. In renal medullary cells, which are chronically exposed to hypertonic stress, both plasma membrane transporters for osmolytes (e.g. betaine, myo-inositol, and taurine) and intracellular enzymes for synthesis of other osmolytes (principally sorbitol and glycerophosphorylcholine) are transcribed and synthesized. Upregulation of a heat shock protein (HSP70) helps to protect cells from the damaging effects of high concentrations of urea [4]. These processes
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