Abstract
The plasma membrane H+-ATPase was purified from the yeast K. lactis. The oligomeric state of the H+-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H+-ATPase hexamer in both MASs as the sole/main oligomeric state—in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H+-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H+-ATPase. It is concluded that the quaternary structure of the H+-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.
Highlights
The yeast plasma membrane H+ -ATPase (Pma1; E.C. 3.6.1.35), a member of the P-type ATPase family [1], pumps protons out of the cell, generating a proton electrochemical gradient that is used as the driving force to transport ions and metabolites into the cell [1]
The H+ -ATPase was found in a large aggregated state that disassembled to two relatively stable macromolecular assembly states (MASs) of different sizes formed by different structural and functional hexamers
Aggregation seems to be canonical for most integral plasma membrane proteins [16]; the self-segregation of membrane proteins, forming stable patches establishing or not a pattern like networks, was recently demonstrated [16]
Summary
The yeast plasma membrane H+ -ATPase (Pma; E.C. 3.6.1.35), a member of the P-type ATPase family [1], pumps protons out of the cell, generating a proton electrochemical gradient that is used as the driving force to transport ions and metabolites into the cell [1]. The plasma membrane H+ -ATPase quaternary structure is controversial. In the plant plasma membrane H+ -ATPase, the enzyme was observed to be in the hexameric state when in the activated state [13]. It was reported that the yeast H+ -ATPase does segregate upon activation by glucose, forming network-like micro-domains in the plasma membrane [15,16]. The isolated MASs displayed significant differences in structure, catalysis, and affinity to nucleotides (ATP and ADP) as revealed by intrinsic fluorescence spectra and enzyme kinetics assay, where the aggregated H+ -ATPase hexamers forming the largest. The hexamer is proposed as the oligomeric state of H+ -ATPase in the yeast plasma membrane, while enzyme activation seems to change its aggregation state
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