Membrane insertion of betaine/GABA transporter during hypertonic stress correlates with nuclear accumulation of TonEBP.
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.
Kempson
Stephen A
SA
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, 46202-5120, USA. skempson@iupui.edu
Beck
Jeffrey A
JA
Lammers
Philip E
PE
Gens
J Scott
JS
Montrose
Marshall H
MH
D000818:Animals; D001622:Betaine / Q000737:chemistry*; D001692:Biological Transport; D019921:Biotinylation; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D050485:GABA Plasma Membrane Transport Proteins; D005765:Gastrointestinal Agents / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012044:Regression Analysis; D012964:Sodium / Q000737:chemistry; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D005765:Gastrointestinal Agents; D007976:Leupeptins; D026901:Membrane Transport Proteins; D011500:Protein Synthesis Inhibitors; D049452:Green Fluorescent Proteins; D001622:Betaine; D003513:Cycloheximide; D012964:Sodium; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde