Characterization of a component of the proton channel of the vacuolar ATPase

The vacuolar H{dollar}sp+{dollar}-ATPase from Neurospora crassa is a large, complex enzyme that pumps protons across the vacuolar membrane. It is composed of a peripheral sector containing 4-5 different subunits and a membrane-bound sector in which the major component is a 16 kDa hydrophobic polypeptide. Because of its hydrophobic properties, it can be extracted into organic solvents, therefore it is referred to as the proteolipid. My goal was to gain a better understanding of the structural and biochemical properties of the 16 kDa polypeptide, and assess what characteristics of the polypeptide may explain its role in proton transport.; I identified the vacuolar proteolipid as a subunit of the V-ATPase by its ability to bind {dollar}sp{lcub}14{rcub}{dollar}C-DCCD. I further isolated it by extraction into organic solvents and purified it by HPLC.; The gene for proteolipid was isolated and sequenced, and the deduced amino acid sequence compared with that of N. crassa mitochondrial proteolipid as well as yeast and bovine chromaffin granule vacuolar-proteolipids. A model for the secondary structure of the vacuolar proteolipid has been proposed based on comparisons with the extensively studied E. coli and mitochondrial proteolipids.; The location of the gene and the size and abundance of transcripts was also determined. The gene for the proteolipid (vma-3) is located close to the gene for the 70 kDa subunit of the vacuolar ATPase. Northern blot analysis revealed 2 mRNA's for the proteolipid. The abundance is similar to that of the other subunits. The smaller of the 2 messages is more abundant.; Finally, I discovered a cDNA that appeared to have 2 mRNA's fused together--the vacuolar proteolipid and a gene homologous to the yeast ILV-5 gene. The sequencing and analysis of this gene is described.