Abstract
In addition to heme’s role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.
| Original language | English |
|---|---|
| Article number | e2104008118 |
| Number of pages | 9 |
| Journal | Proceedings of the National Academy of Sciences |
| Volume | 118 |
| Issue number | 22 |
| Early online date | 25 May 2021 |
| DOIs | |
| Publication status | Published - 1 Jun 2021 |
Bibliographical note
Funding Information:ACKNOWLEDGMENTS. We would like to acknowledge Professor David Stephens (Department of Biochemistry, University of Bristol) for support in cell culturing and helpful discussions. We thank Dipti Vashi and Sharon Munday in the Protein Expression Laboratory (PROTEX-University of Leicester) for preparing the expression clones. We gratefully acknowledge the Wolfson Bioimaging Facility for their support and assistance in this work and BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (Grant Number: L01386X) for funding the FLIM microscope. The research was supported by the Leverhulme Trust (RPG-2016-397) awarded to A.J.H. and E.L.R.; G.C.-H.L. is grateful to the College of Science and Engineering, University of Leicester for a PhD studentship; and A.E.G. is grateful to the Engineering and Physical Sciences Research Council for a PhD studentship.
Funding Information:
We would like to acknowledge Professor David Stephens (Department of Biochemistry, University of Bristol) for support in cell culturing and helpful discussions. We thank Dipti Vashi and Sharon Munday in the Protein Expression Laboratory (PROTEX-University of Leicester) for preparing the expression clones. We gratefully acknowledge the Wolfson Bioimaging Facility for their support and assistance in this work and BrisSynBio, a BBSRC/EPSRC-funded Synthetic Biology Research Centre (Grant Number: L01386X) for funding the FLIMmicroscope. The research was supported by the Leverhulme Trust (RPG-2016- 397) awarded to A.J.H. and E.L.R.; G.C.-H.L. is grateful to the College of Science and Engineering, University of Leicester for a PhD studentship; and A.E.G. is grateful to the Engineering and Physical Sciences Research Council for a PhD studentship.
Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.
Keywords
- Heme
- cell signaling
- regulation
- fluorescence lifetime
- imaging