Figure 4. SUMOylation is greatly accelerated in the droplet phase.

(A) Schematic of workflow to measure SUMOylation rate in the droplet phase. Total SUMOylation rate is measured by simply mixing all components and adding ATP. Bulk rate is measured by centrifuging the total mixture prior to addition of ATP to sediment droplets, transferring the supernatant to a new tube, and then initiating the reaction with ATP. The difference between Total and Bulk rate yields the Droplet rate.

(B) Representative plot showing the production of SUMOylated RanGAP* over time in the total (+rap), bulk (+rap), and total (+DMSO) solutions. Error bars on total (+rap) and bulk (+rap) represent the SEM of 3 experiments.

(C) Schematic depicting the two approaches used to calculate droplet volume fraction. Top panel shows the equation used based on conservation of mass. [ ] represent concentrations in the indicated phase measured by fluorescence imaging. Bottom panel illustrates the direct measurement approach based on confocal imaging of a three-dimensional volume.

(D) Volume-normalized rate toward RanGAP* for the droplet, droplet equivalent concentration (DEC), and bulk phases. Error bars represent the SEM from 6 independent experiments. The statistical significance was assessed by a two-tailed, unpaired Student’s t-test. ** represents a p-value < 0.01 (0.0022), and **** represents a p-value < 0.0001 (1.4 × 10⁻⁶). Droplet rate was determined from the difference between the average total reaction rate and average bulk reaction rate, with errors propagated accordingly.

Panels B and D have associated raw data.