Versatile Labeling and Detection of Endogenous Proteins Using Tag-Assisted Split Enzyme Complementation

Makhija, Suraj; Brown, David; Rudlaff, Rachel M.; Doh, Julia K.; Bourke, Struan; Wang, Yina; Zhou, Shuqin; Cheloor-Kovilakam, R.

doi:10.1021/acschembio.0c00925

Cited by 14 publications

(15 citation statements)

References 49 publications

(104 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fluorescence cross-correlation spectroscopy (FCCS) can detect bulk molecular interactions, yet it does not provide spatial trajectories for individual molecules, which are useful for measuring such properties as chromatin residence time and anomalous diffusion ( Hansen et al, 2020 ; Hansen et al, 2017 ; Izeddin et al, 2014 ; McSwiggen et al, 2019 ; Nguyen et al, 2021 ). Bimolecular fluorescence complementation (BiFC) detects molecular interactions based on the reconstitution of a fluorescent protein or HaloTag from two split halves fused to interacting partners ( Ghosh et al, 2000 ; Hu et al, 2002 ; Kerppola, 2008 ; Makhija et al, 2021 ; Shao et al, 2021 ). While BiFC can be combined with single-molecule imaging ( Mao et al, 2021 ; Nickerson et al, 2014 ; Shao et al, 2021 ), a drawback of this approach is that the extremely strong association of split proteins perturbs the binding equilibrium of their interacting partners ( Kerppola, 2008 ; Kodama and Hu, 2012 ; Nickerson et al, 2014 ), making it impossible to accurately measure dynamic interactions.…”

Section: Introductionmentioning

confidence: 99%

Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)

Graham

Ferrie

Dailey

et al. 2022

eLife

View full text Add to dashboard Cite

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a sub-population of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

show abstract

Section: Introductionmentioning

confidence: 99%

Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)

Graham

Ferrie

Dailey

et al. 2022

eLife

View full text Add to dashboard Cite

show abstract

“…To visualize Csf1 and Hob2, we initially tagged them endogenously with GFP on their C-termini, but the signal was low (not shown). Instead of using a single GFP, we endogenously tagged the proteins with seven copies of GFP11, a fragment of GFP that will self-assemble with the remainder of the protein, GFP1–10 ( Makhija et al, 2021 ). The tagged proteins are functional ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Vps13-like proteins provide phosphatidylethanolamine for GPI anchor synthesis in the ER

Toulmay

Whittle

Yang

et al. 2022

Journal of Cell Biology

View full text Add to dashboard Cite

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kučinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis.

show abstract

“…Most endogenous proteins exist at much lower concentrations in cells relative to overexpressed proteins, which can make both fluorescence selection and structural analysis of these relatively rare targets challenging. For selection, it has been estimated that ∼50% of the human proteome is expressed above the FACS detection limit using mNG2[11] tagging (14), and signal amplification using our recent tag-assisted split enzyme complementation method ( 29 ) could further expand the accessible range. For purification, the recent development of affinity grids offers another potential solution to the study of low-abundance targets ( 30 , 31 ).…”

Section: Discussionmentioning

confidence: 99%

Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins

Zhao

Makhija

Zhou³

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

show abstract

Versatile Labeling and Detection of Endogenous Proteins Using Tag-Assisted Split Enzyme Complementation

Cited by 14 publications

References 49 publications

Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)

Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)

Vps13-like proteins provide phosphatidylethanolamine for GPI anchor synthesis in the ER

Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins

Contact Info

Product

Resources

About