Structural studies of the vacuolar H+-pyrophosphatase: Sequence analysis and identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide

Abstract

We determined the amino acid residues of the H+-translocating inorganic pyrophosphatase (H+-PPase) of pumpkin which are covalently labeled by two fluorescent labeling reagents; N-cyclohexyI-N'-[4-(dimethyl amino)-a-naphthyl] carbodiimide (NCD) and N-pyrenylmaleimide (NPM). NCD and NPM are fluorescent analogues of N,N'-dicycrohexylcarbodiimide and N-ethylmaleimide, respectively, and inactivate H+-PPase activity. Excess Mg2+ protected the H+-PPase from the inactivation by these reagents. Furthermore, we identified the cDNA clone encoding the pumpkin H+-PPase in order to determine the position of labeled residues. The nucleotide sequence of the cDNA clone contains a 2,304 bp open reading frame encoding a polypeptide with 768 amino acids. Chemical sequence analysis of fluorescent peptide fragments revealed that Glu749 located in the C-terminal putative transmembrane a-helix was a NCD-labeled residue, and Cys632 was a NPM-labeled residue located in a putative cytosolic domain. The amino acid sequence of the region that includes Glu749 is highly conserved in H+-PPases from other plants and it also shows some sequence similarity with the region of the carbodiimide-reactive Glu (or Asp) of F0F1-ATPase c-subunit. The reactive glutamic acids in these proteins are located at the last C-terminal transmembrane a-helix. We also found that the H+-PPase shows significant amino acid sequence similarity to Kdp-ATPase A chain of E. coli. This similarity between the two different proteins suggest that they have evolved from a common ancestor and may utilize a common basic mechanism for ion transport.