Abstract
The S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.
Highlights
The S. cerevisiae plasma membrane H+-ATPase, Pma[1], is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma[1] (MCP)
We found that TMH1, 2, and 4B moved, but TMH3, 4 A, and 5–10 were largely stationary among these structures
Based on our surprising observation that the Pma[1] hexamer encloses ~57 ordered lipids, we suggest that this protein-lipid complex structure is the major building block of the S. cerevisiae membrane compartment of Pma1 (MCP)
Summary
The S. cerevisiae plasma membrane H+-ATPase, Pma[1], is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma[1] (MCP). Like other plasma membrane H+-ATPases, Pma[1] assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases It has been unclear how Pma[1] organizes the yeast membrane into MCP microdomains, or why it is that Pma[1] needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. During the preparation of this manuscript, a 3.3 Å resolution cryo-EM structure of the Neurospora crassa Pma[1] was posted in BioRxiv[35] Despite such progress, how the yeast Pma[1] interacts with membrane lipids to organize the MCP microdomains and how Pma[1] switches between the autoinhibited state and the active state remain unknown. We report a high-resolution cryo-EM study of autoinhibited and activated native Pma[1] hexamers embedded in endogenous lipids, which sheds light on these long-standing questions
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