A Hexameric Form of the Neurospora Crassa Plasma Membrane H+-ATPase

As isolated by our recently developed large-scale procedure, the Neurospora plasma membrane H+-ATPase exists as a homogeneous, oligomeric complex of 105,000-Da monomers with a molecular mass equivalent to a spherical protein of about 1 million Da, as judged by its behavior during chromatography on calibrated columns of Sepharose CL-6B and CL-4B. Treatment of this complex with the nonionic detergent, Tween 20, followed by Sepharose column chromatography in the presence of this detergent produces particles with an apparent molecular mass reduced by 100-300 kDa, and, importantly, when the isolated complex is treated with Tween 20 and then subjected to Sepharose chromatography in the absence of detergent, fully viable, largely detergent-free, homogeneous particles with a molecular mass equivalent to a spherical protein of 670,000 Da are formed. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, treatment of the particles isolated in the presence of Tween 20 with glutaraldehyde progressively yields dimers, trimers, tetramers, pentamers, and hexamers of the 105,000-Da monomer, with the expected precursor-product relationships, but no species larger than a hexamer is formed. These results thus strongly indicate that these particles are hexamers of 105,000-Da monomers. Glutaraldehyde crosslinking experiments with the ca. 1 million- and 670,000-Da particles indicate that they too are hexamers, suggesting that the differences in the apparent sizes of the three types of particles are most likely due to bound detergents. Possible implications of these findings are discussed.