Isolation of native plasma membrane H+-ATPase (Pma1p) in both the active and basal activation states

Jesper Torbøl Pedersen*, Tamara Kanashova, Gunnar Dittmar, Michael Palmgren

*Corresponding author for this work

    Research output: Contribution to journalArticleResearchpeer-review

    8 Citations (Scopus)

    Abstract

    The yeast plasma membrane H+-ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H+-ATPases are autoinhibited.

    Original languageEnglish
    Pages (from-to)774-783
    Number of pages10
    JournalFEBS Open Bio
    Volume8
    Issue number5
    DOIs
    Publication statusPublished - May 2018

    Keywords

    • P-type ATPase
    • Pma1p
    • acetylated tubulin
    • autoinhibitory regulation

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