Membrane-mediated dimerization potentiates PIP5K lipid kinase activity
Abstract
The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2 containing membranes and dimerize in a protein density dependent manner. Although dispensable for cooperative PI(4,5)P2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane bound kinase.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for all figures.
Article and author information
Author details
Funding
National Science Foundation (CAREER MCB-2048060)
- Scott D Hansen
University of Oregon, Department of Chemistry and Biochemistry (lab startup funds)
- Scott D Hansen
Novo Nordisk Foundation Challenge Programme (Center for Geometrically Engineered Cellular Systems)
- Albert A Lee
- Jay T Groves
National Institute of Health, NIGMS (National Research Service Award (NRSA),F32 GM111010-02)
- Scott D Hansen
National Institute of Health, NIGMS (T32 GM007759)
- Benjamin R Duewell
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Janice L Robertson, Washington University in St Louis, United States
Version history
- Received: September 9, 2021
- Preprint posted: September 22, 2021 (view preprint)
- Accepted: August 16, 2022
- Accepted Manuscript published: August 17, 2022 (version 1)
- Version of Record published: September 13, 2022 (version 2)
Copyright
© 2022, Hansen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Biochemistry and Chemical Biology
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Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
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