Glyceraldehyde 3-phosphate dehydrogenase is required for band 3 (anion exchanger 1) membrane residency in the mammalian kidney.
The mammalian kidney isoform of the essential chloride-bicarbonate exchanger AE1 differs from its erythrocyte counterpart, being shorter at its N terminus. It has previously been reported that the glycolytic enzyme GAPDH interacts only with erythrocyte AE1, by binding to the portion not found in the kidney isoform. (Chu H, Low PS. Biochem J 400:143-151, 2006). We have identified GAPDH as a candidate binding partner for the C terminus of both AE1 and AE2. We show that full-length AE1 and GAPDH coimmunoprecipitated from both human and rat kidney as well as from Madin-Darby canine kidney (MDCK) cells stably expressing kidney AE1, while in human liver, AE2 coprecipitated with GAPDH. ELISA and glutathione S-transferase (GST) pull-down assays using GST-tagged C-terminal AE1 fusion protein confirmed that the interaction is direct; fluorescence titration revealed saturable binding kinetics with Kd 2.3±0.2 μM. Further GST precipitation assays demonstrated that the D902EY residues in the D902EYDE motif located within the C terminus of AE1 are important for GAPDH binding. In vitro GAPDH activity was unaffected by C-terminal AE1 binding, unlike in erythrocytes. Also, differently from red cell N-terminal binding, GAPDH-AE1 C-terminal binding was not disrupted by phosphorylation of AE1 in kidney AE1-expressing MDCK cells. Importantly, small interfering RNA knockdown of GAPDH in these cells resulted in significant intracellular retention of AE1, with a concomitant reduction in AE1 at the cell membrane. These results indicate differences between kidney and erythrocyte AE1/GAPDH behavior and show that in the kidney, GAPDH is required for kidney AE1 to achieve stable basolateral residency.
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Department of Medical Genetics, University of Cambridge, and Cambridge Institute for Medical Research, Addenbrooke's Hospital, Box 139, Hills Rd., Cambridge CB2 0XY, UK.
Blake-Palmer
Katherine G
KG
Fry
Andrew C
AC
Best
Alison
A
Brown
Alice C N
AC
Hiemstra
Thomas F
TF
Horita
Shoko
S
Zhou
Aiwu
A
Toye
Ashley M
AM
Karet
Fiona E
FE
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D027321:Anion Transport Proteins / Q000378:metabolism; D017920:Antiporters / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D064606:SLC4A Proteins
D001457:Anion Exchange Protein 1, Erythrocyte; D027321:Anion Transport Proteins; D017920:Antiporters; D064606:SLC4A Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005982:Glutathione Transferase