Membrane-mediated dimerization potentiates PIP5K lipid kinase activity
- Department of Chemistry and Biochemistry, University of Oregon, United States
- Institute of Molecular Biology, University of Oregon, United States
- Department of Chemistry, University of California, Berkeley, United States
- California Institute for Quantitative Biosciences, United States
- Department of Molecular and Cell Biology, United States
Figures
Figure 1 with 6 supplements
PIP4K and PIP5K can bind to PI(4,5)P2 membranes with distinct oligomeric states.
(A) Cartoon showing the kinase domain orientation and proposed oligomerization states of PIP4K and PIP5K homodimers. PIP5K potentially exists in a monomer–dimer equilibrium, while PIP4K is a …
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Figure 1—source data 1
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Figure 1—source data 2
Rlated to Figure 1D.
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Figure 1—source data 3
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Figure 1—source data 4
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Figure 1—source data 5
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Figure 1—source data 6
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Figure 1—source data 7
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Figure 1—source data 8
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Figure 1—figure supplement 1
Size-exclusion chromatography (SEC) analysis of purified proteins.
(A) SEC elution profile of Sigma molecular weight standards (Cat# 69385) containing thyroglobulin bovine (~670 kDa), γ-globulins from bovine blood (~150 kDa), albumin chicken egg grade VI (~44.3 …
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Figure 1—figure supplement 1—source data 1
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Figure 1—figure supplement 2
mNeonGreen calibration curve for measuring the solution concentration in cell lysate.
(A–C) Plot showing the linear relationship between the concentration of purified mNeonGreen and fluorescence emission intensity of the solution measured using a BioTek 96-well plate reader. Curves …
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Figure 1—figure supplement 2—source data 1
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Figure 1—figure supplement 3
Molecular brightness distribution of Ax488-PIP4KB and Ax488-PIP5KB.
(A) Single-molecule brightness distributions for Sortase-labeled Alexa488-PIP4K and Alexa488-PIP5K bound to supported membranes containing 96% DOPC and 4% PI(4,5)P2.
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Figure 1—figure supplement 3—source data 1
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Figure 1—figure supplement 4
Molecular brightness and photobleaching analysis of mNG-PIP4K and mNG-PIP5K.
(A) Single-molecule total internal reflection fluorescence (TIRF) microscopy images of supported lipid bilayers (SLBs) incubated with either 1 pM mNG-PIP4KB, 5 pM mNG-mPIP5KA, or 5 pM mNG-zPIP5KA. (B…
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Figure 1—figure supplement 4—source data 1
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Figure 1—video 1
Membrane -binding dynamics and multistep photobleaching of mNG-PIP4KB visualized by single-molecule total internal reflection fluorescence (smTIRF) microscopy.
Membrane composition: 96% DOPC, 4% PI(4,5)P2. Video associated with Figure 1. Scale bar is 2 µm.
Figure 1—video 2
Membrane -binding dynamics and single -step photobleaching of mNG-PIP5KB visualized by single-molecule total internal reflection fluorescence (smTIRF) microscopy.
Membrane composition: 96% DOPC, 4% PI(4,5)P2. Video associated with Figure 1. Scale bar is 2 µm.
Figure 2 with 5 supplements
Protein density-dependent changes in PIP5K membrane binding.
(A) Supported lipid bilayer assay for measuring the single-molecule membrane-binding behavior Ax647-PIP5KB at low and high membrane surface densities of PIP5KB. Note that the ‘high-density’ kinase …
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Figure 2—source data 1
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Figure 2—source data 2
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Figure 2—source data 3
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Figure 2—figure supplement 1
Quantification of Alexa647-PIP5KB photobleaching kinetics.
(A) Montage of total internal reflection fluorescence microscopy (TIRF-M) images showing the change in fluorescence of glass-immobilized Ax647-PIP5KB in the absence and presence of oxygen-scavenging …
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Figure 2—figure supplement 1—source data 1
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Figure 2—figure supplement 2
Protein density dependent changes in Ax647-PIP5K single molecule dwell time distributions.
(A) Zoomed graph showing the initial curve fits of the Ax647-PIP5KB dwell time distributions shown in Figure 2B.
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Figure 2—figure supplement 2—source data 1
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Figure 2—figure supplement 3
Calibration of Alexa647-PIP5KB membrane surface density measurements.
(A) Calculation of scaling factor used from comparing the fluorescence of Ax647-PIP5KB and Atto655-DPPE. The fluorescence intensity of small unilamellar vesicles containing varying concentrations of …
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Figure 2—figure supplement 3—source data 1
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Figure 2—video 1
Membrane -binding dynamics of 1 pM Ax647-PIP5KB visualized by single-molecule total internal reflection fluorescence (smTIRF) microscopy.
Membrane composition: 96% DOPC, 4% PI(4,5)P2. Video associated with Figure 2B. Scale bar is 2 µm.
Figure 2—video 2
Membrane -binding dynamics of 1 pM Ax647-PIP5KB plus 50 nM dark PIP5KB visualized by single-molecule total internal reflection fluorescence (smTIRF) microscopy.
Membrane composition: 96% DOPC, 4% PI(4,5)P2. Video associated with Figure 2B. Scale bar is 2 µm.
Figure 3 with 1 supplement
PIP5K binds cooperatively to PI(4,5)P2 independent of dimerization.
(A) Kinase domain orientation for the zebrafish PIP5KA homodimer (4TZ7.pdb). Salt bridges formed between Asp and Arg side chains are colored and shown in zoomed images. (B) Sequence alignment …
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Figure 3—source data 1
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Figure 3—source data 2
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Figure 3—source data 3
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Figure 3—source data 4
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Figure 3—figure supplement 1
Dimer interface mutation does not alter diffusion of Ax647-PIP5K at low protein densities.
(A, B) Step-size distributions are indistinguishable when measured in the presence of (A) 5 pM Ax647-PIP5KB (0.171 ± 0.006 µm2/s, N = 174,748 steps) or (B) 5 pM Ax647-PIP5KB (D51R) (0.174 ± 0.001 µm2…
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Figure 3—figure supplement 1—source data 1
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Figure 4 with 2 supplements
Direct visualization of mNG-PIP5KB membrane-mediated dimerization.
(A) Size-exclusion chromatography (SEC) elution profiles of purified mNG-PIP5KB and mNG-PIP5KB (D51R). The monomeric molecular weight of each kinase equals 75 kDa. Protein was at a concentration of …
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Figure 4—source data 1
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Figure 4—source data 2
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Figure 4—source data 3
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Figure 4—source data 4
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Figure 4—source data 5
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Figure 4—source data 6
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Figure 4—source data 7
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Figure 4—figure supplement 1
Quantification of PIP5K dimerization-based molecular brightness.
(A–D) Molecular brightness distributions measured in the presence of 100 pM mNG-mPIP5KA (A), mNG-mPIP5KA (D92R) (B), mNG-zPIP5KA (C), or mNG-zPIP5KA (D84R) (D) using diluted cell lysate. A threshold …
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Figure 4—figure supplement 1—source data 1
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Figure 4—video 1
Direct visualization of membrane-mediated dimerization of mNG-PIP5KB with single -molecule resolution.
Membrane composition: 96% DOPC, 4% PI(4,5)P2. Video associated with Figure 4J. Scale bar is 1 µm.
Figure 5 with 2 supplements
Membrane-mediated dimerization potentiates PIP5K lipid kinase activity.
(A–C) Representative kinetic traces monitoring the production of PI(4,5)P2 in the presence of 1–5 nM PIP5KB, PIP5KB (D51R), or PIP5KB (D51R/R254D). The PI(4,5)P2 membrane surface density was …
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Figure 5—source data 1
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Figure 5—source data 2
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Figure 5—source data 3
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Figure 5—source data 4
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Figure 5—source data 5
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Figure 5—source data 6
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Figure 5—source data 7
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Figure 5—figure supplement 1
Dimerization enhances PIP5KA lipid kinase activity.
(A, B) Kinetics of PI(4,5)P2 production measured in the presence of either mouse PIP5KA or PIP5KA (D92R). The production of PI(4,5)P2 was monitored by the presence of 20 nM Alexa488-PLCδ. The …
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Figure 5—figure supplement 1—source data 1
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Figure 5—figure supplement 2
Fluorescence correlation spectroscopy membrane density calibration.
(A) Autocorrelation of his10-mNeonGreen (his10-mNG) measured by fluorescence correlation spectroscopy (FCS). Membrane composition: 98% DOPC, 2% NiNTA-DGS. (B, C) Calibration of mNG-PIP5KB and …
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Figure 5—figure supplement 2—source data 1
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Figure 6 with 2 supplements
PIP5K dimerization stabilizes phosphatidylinositol phosphate (PIP) compositional patterns.
(A) Diagram showing the network architecture of the bistable kinase–phosphatase-competitive reaction. The positive feedback loop of the 5-phosphatase, DrrA-OCRL, is regulated by product binding (Hans…
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Figure 6—source data 1
Related to Figure 6D.
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Figure 6—video 1
Visualization of mNG-PIP5KB localization during phosphatidylinositol phosphate (PIP) compositional pattern formation.
Reaction reconstituted in the presence of 50 nM mNG-PIP5KB, 20 nM DrrA-OCRL, and 20 nM Cy3-PLCδ. Initial membrane composition: 96% DOPC, 2% PI(4)P, 2% PI(4,5)P2. Video associated with Figure 6C. …
Figure 6—video 2
Visualization of mNG-PIP5KB (D51R) localization during phosphatidylinositol phosphate (PIP) compositional pattern formation.
Reaction reconstituted in the presence of 1 µM mNG-PIP5KB (D51R), 20 nM DrrA-OCRL, and 20 nM Cy3-PLCδ. Initial membrane composition: 96% DOPC, 2% PI(4)P, 2% PI(4,5)P2. Video associated with Figure 6C…
Figure 7 with 2 supplements
Reaction trajectory variation based on stochastic membrane-mediated dimerization.
(A) Lipid phosphorylation reactions reconstituted in 5 µm × 5 µm chromium-patterned-supported membranes in the presence of 5 nM PIP5K and 10 nM PIP5K (D51R). Reactions were visualized using 20 nM …
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Figure 7—source data 1
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Figure 7—source data 2
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Figure 7—source data 3
Related to Figure 7E.
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Figure 7—video 1
Visualization of lipid phosphorylation reactions reconstituted in 5 µm x 5 µm chromium -patterned -supported membranes.
Reaction contains 5 nM PIP5KB and 20 nM Alexa488-PLCδ. Video associated with Figure 7A. Initial membrane composition: 96% DOPC, 4% PI(4)P. Scale bar is 10 µm.
Figure 7—video 2
Visualization of lipid phosphorylation reactions reconstituted in 5 µm x 5 µm chromium -patterned -supported membranes.
Reaction contains 10 nM PIP5KB (D51R) and 20 nM Alexa488-PLCδ. Video associated with Figure 7A. Initial membrane composition: 96% DOPC, 4% PI(4)P. Scale bar is 10 µm.
Figure 8
Model for PIP5K membrane-mediated dimerization.
Mediated by electrostatic interactions, PIP5K can associate with membranes as a monomer. The specificity loop stabilizes membrane association and enables PIP5K to catalyze the phosphorylation of …
Tables
Table 1
Protein density dependent changes in Ax647-PIP5KB membrane binding behavior.
| Protein visualized | [5KB] | τ1 ± SD (s) | τ2± SD (s) | α ± SD | N | D1 ± SD (µm2/s) | D2 ± SD (µm2/s) | α ± SD | Steps |
|---|---|---|---|---|---|---|---|---|---|
| 1 pM Ax647-5KB | 0 | 0.453 ± 0.013 | – | – | 7018 | 0.148 ± 0.005 | – | – | 66,889 |
| 1 pM Ax647-5KB | 0.2 nM | 0.475 ± 0.037 | – | – | 4855 | 0.058 ± 0.011 | 0.194 ± 0.012 | 0.20 ± 0.06 | 48,888 |
| 1 pM Ax647-5KB | 1 nM | 0.559 ± 0.009 | – | – | 4783 | 0.038 ± 0.001 | 0.189 ± 0.004 | 0.27 ± 0.02 | 56,027 |
| 1 pM Ax647-5KB | 5 nM | 0.599 ± 0.055 | 1.354 ± 0.32 | 0.72 ± 0.17 | 4268 | 0.038 ± 0.002 | 0.173 ± 0.007 | 0.40 ± 0.04 | 66,680 |
| 1 pM Ax647-5KB | 10 nM | 0.895 ± 0.032 | 2.791 ± 0.27 | 0.94 ± 0.03 | 2362 | 0.039 ± 0.004 | 0.164 ± 0.002 | 0.48 ± 0.05 | 47,103 |
| 1 pM Ax647-5KB | 50 nM | 0.787 ± 0.057 | 2.08 ± 0.078 | 0.70 ± 0.03 | 1530 | 0.043 ± 0.002 | 0.142 ± 0.008 | 0.63 ± 0.03 | 35,678 |
| 1 pM Ax647-4KB | 0 | 0.021 ± 0.005 | 0.058 ± 0.007 | 0.39 ± 0.15 | 227,275 |
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SD, standard deviation from 3 to 5 technical replicates; N, total number of molecules tracked in 3–5 technical replicates; steps, total number of particle displacements measured in 3–5 technical replicates; alpha, fraction of molecules that have the characteristic dwell time or diffusion coefficient (τ1 or D1), τbleaching, Ax647-5KB = 26.7 s (see Figure 2—figure supplement 1); membrane composition, 98% DOPC, 2% PI(4,5)P2.
Table 2
Dimerization dependent changes in Ax647-PIP5KB membrane binding behavior.
| Protein visualized | [5KB] | % PIP2 | τ1 ± SD (s) | τ2 ± SD (s) | α ± SD | N | D1 ± SD (µm2/s) | D2 ± SD (µm2/s) | α ± SD | Steps |
|---|---|---|---|---|---|---|---|---|---|---|
| 25 pM Ax647-5KB | 0 | 1 | 0.096 ± 0.05 | – | – | 3422 | – | – | – | – |
| 5 pM Ax647-5KB | 0 | 2 | 0.64 ± 0.092 | – | – | 4525 | 0.171 ± 0.006 | – | – | 174,748 |
| 2 pM Ax647-5KB | 0 | 4 | 0.74 ± 0.088 | 3.43 ± 0.29 | 0.59 ± 0.06 | 1920 | – | – | – | – |
| 25 pM Ax647-5KB (D51R) | 0 | 1 | 0.090 ± 0.01 | – | – | 4848 | – | – | – | – |
| 5 pM Ax647-5KB (D51R) | 0 | 2 | 0.649 ± 0.043 | – | – | 2542 | 0.174 ± 0.001 | – | – | 238,548 |
| 2 pM Ax647-5KB (D51R) | 0 | 4 | 0.864 ± 0.077 | 3.97 ± 0.37 | 0.58 ± 0.03 | 1850 | – | – | – | – |
| 1 pM Ax647-5KB | 50 nM | 2 | 0.709 ± 0.24 | 2.49 ± 0.27 | 0.53 ± 0.08 | 9693 | 0.022 ± 0.002 | 0.067 ± 0.005 | 0.50 ± 0.07 | 311,198 |
| 1 pM Ax647-5KB (D15R) | 50 nM | 2 | 0.416 ± 0.02 | – | – | 8218 | 0.133 ± 0.008 | – | – | 74,928 |
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SD, standard deviation from 3 to 5 technical replicates; N, total number of molecules tracked in 3–5 technical replicates; steps, total number of particle displacements measured in 3–5 technical replicates; alpha, fraction of molecules that have the characteristic dwell time or diffusion coefficient (τ1 or D1), τbleaching, Ax647-5KB = 26.7 s (see Figure 2—figure supplement 1); membrane composition, 96–99% DOPC, 1–4% PI(4,5)P2.
Additional files
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Transparent reporting form
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Supplementary file 1
Plasmids and recombinant proteins.
Description of plasmids used for recombinant protein expression, purification, and transient transfection.
- https://cdn.elifesciences.org/articles/73747/elife-73747-supp1-v2.docx
