Trafficking of renal betaine/GABA transporter (BGT1) is disrupted by mutation at T40

Abstract

Cells in renal medulla accumulate several osmolytes to survive hypertonicity and this is reproduced in renal MDCK cells. In response to hypertonic stress (Hyp, 500 mOsm) for 24 h there is upregulation of BGT1 mRNA and protein synthesis followed by trafficking to the plasma membrane (PM) and a marked increase in betaine transport. PM trafficking motifs reside near the C‐terminus of BGT1 and truncation at K560 resulted in a protein that remained intracellular during Hyp stress, as expected. The K560Δ mutant expressed in MDCK cells was colocalized with an ER marker but not markers for endosomes or Golgi. Surprisingly, substitution of A at T40 of native BGT1, to block phosphorylation, also prevented trafficking to the PM during Hyp stress. The T40A mutant was not retained in the ER and colocalized with markers for Golgi and endosomes suggesting, indirectly, that it was not misfolded. Mutation of T40 to E or D, to mimic phosphorylation, restored normal trafficking to the PM. HEK cells transfected with K560Δ or T40A mutants had 10% of GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Data suggest that normal BGT1 trafficking (or PM retention) requires both C‐terminal motifs and phosphorylation at T40.