MDCK cells stably transfected with betaine/GABA transporter tagged with EGFP (EGFP-BGT) were used to study plasma membrane insertion of EGFP-BGT. Adaptive response to hypertonicity requires nuclear migration of TonEBP. Confocal microscopy showed that after 6 h hypertonicity, the nuclear/cytoplasmic ratio of TonEBP fluorescence was increased to 2.4 compared to 1.4 in isotonic controls (P<0.001). The ratio in hypertonic cells was reduced by the proteasome inhibitor MG-132 in a dose-dependent way. Inhibition was 50% at 3 microM. After 6 h, hypertonicity expressed EGFP-BGT was localized in the plasma membrane, but there was no change in total EGFP-BGT abundance compared to isotonic controls. In contrast, EGFP-BGT remained mostly intracellular when 3 microM MG-132 was included in the hypertonic medium. The transport function of EGFP-BGT was studied as Na(+)-dependent uptake of [(3)H]GABA. This was not changed by MG-132 in isotonic controls, but MG-132 produced dose-dependent inhibition of hypertonic upregulation of Na(+)/GABA cotransport. Inhibition was 80% at 3 muM MG-132. Transport likely reflects membrane insertion of EGFP-BGT and there was a positive correlation (P<0.05) between Na(+)/GABA cotransport and the N/C ratio of TonEBP. Results are consistent with a role for TonEBP-mediated transcription in synthesis of additional proteins required for membrane insertion of EGFP-BGT protein.