Subcellular redistribution of the renal betaine transporter during hypertonic stress.
The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.
Kempson
Stephen A
SA
Department of Cellular and Integrative Medicine, Indiana University School of Medicine, Medical Sciences 451, 635 Barnhill Drive, Indianapolis, IN 46202-5120, USA. skempson@iupui.edu
Parikh
Vaibhave
V
Xi
Lixuan
L
Chu
Shaoyou
S
Montrose
Marshall H
MH
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D050485:GABA Plasma Membrane Transport Proteins; D006982:Hypertonic Solutions; D007668:Kidney / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D051379:Mice; D008809:Mice, Inbred C3H; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013395:Sucrose / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D006982:Hypertonic Solutions; C072207:betaine plasma membrane transport proteins; D005680:gamma-Aminobutyric Acid; D013395:Sucrose