Deletion of seven amino acid residues from the gamma subunit of Escherichia coli H+-ATPase causes total loss of F1 assembly on membranes

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Escherichia coli mutant NR70 possesses a defective H+-translocating ATPase (ATP synthase).
• The gamma subunit is a critical component of the ATP synthase complex.

Purpose of the Study:

• To clone and characterize the mutant gene for the gamma subunit of H+-translocating ATPase from Escherichia coli NR70.
• To investigate the structural and functional consequences of the mutation on ATP synthase assembly.

Main Methods:

• Gene cloning and nucleotide sequencing of the mutant gamma subunit gene.
• Analysis of amino acid sequence and hydropathic character.
• Assessment of F1 subunit assembly on the F0 portion of the mutant enzyme.

Main Results:

• A 21-base pair deletion in the gamma subunit gene resulted in the loss of seven amino acids and a hydrophobic domain.
• The deletion in the gamma subunit impaired the assembly of F1 subunits onto the F0 portion.
• Introduction of the mutant allele into a wild-type strain did not confer neomycin resistance, suggesting the phenotype is unrelated to the gamma subunit defect.

Conclusions:

• The deleted hydrophobic domain of the gamma subunit is essential for the proper assembly of the F1 portion of H+-translocating ATPase.
• The neomycin-resistant phenotype observed in some studies is not directly associated with the identified defect in the gamma subunit of NR70.