Identification of zeta-crystallin/NADPH : quinone reductase as a renal glutaminase mRNA pH response element-binding protein

Increased renal ammoniagenesis and bicarbonate synthesis from glutamine dur ing chronic metabolic acidosis facilitate the excretion of acids and partia lly restore normal acid-base balance. This adaptation is sustained, in part , by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis of the mitochondrial glutaminase, A direct repeat of a n 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA binds a unique protein with high affinity and specificity. Expression of various chimeric mRNAs in LLC-PK1-FBPase(+) cells demonstrated that a si ngle 8-base AU sequence is both necessary and sufficient to function as a p H response element (pH RE). A biotinylated oligoribonucleotide containing t he direct repeat was used as an affinity ligand to purify the pH RE-binding protein from a cytosolic extract of rat renal cortex. The purified binding activity retained the same specific binding properties as observed with cr ude extracts and correlated with the elution of a 36-kDa protein. Microsequ encing by mass spectroscopy and Western blot analysis were used to identify this protein as zeta -crystallin/NADPH:quinone reductase, The purified pro tein contained eight tryptic peptides that were identical to sequences foun d in mouse zeta -crystallin and three peptides that differed by only a sing le amino acid. The observed differences may represent substitutions found i n the rat homolog. A second protein purified by this protocol was identifie d as T-cell-restricted intracellular antigen-related protein (TIAR), Howeve r, the purified TIAR neither bound nor affected the binding of zeta -crysta llin/NADPH:quinone reductase to the pH RE. Further; more, specific antibodi es to zeta -crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified pr otein. Thus, zeta -crystallin/NADPH:quinone reductase is a pH response elem ent-binding protein.