Figure 2.
Figure 2. Peroxisome protein import in Xenopus egg extract. (A) mScarlet-SKL (0.6 µM) was added at time point zero to cleared Xenopus egg extract. Where indicated, 5 or 10 mM ATPγS was added 20 min before substrate. The samples were immediately mounted on PEG-passivated glass coverslips and imaged at the indicated time points, using a spinning-disk confocal microscope. The mean fluorescence of peroxisomes was determined from >10 images using an automated image analysis script. Shown are the combined data of two different experiments (>20 images per time point), each normalized to the final time point of the control. For each time point, the mean and the standard deviation of the mean are given. (B) Cleared extract was incubated with 0.5 µM GFP-SKL for 1 h at 20–23°C and imaged with a spinning-disk confocal microscope. (C) As in B but at the end of the incubation, 0.75 µM GFP-fluorescence quenching nanobodies (Kirchhofer et al., 2010) were added for ∼15 min before imaging. (D) Quantification of the mean peroxisome fluorescence in B and C of two separate import reactions. Shown are the mean and standard deviation of >20 images. (E) As in B, but with GFP lacking the SKL sequence. (F) As in C, but with GFP lacking the SKL sequence. (G) Quantification of the total fluorescence in E and F of two separate import reactions. Shown are the mean and standard deviation of >20 images. (H) mScarlet-SKL (0.6 µM) was incubated with cleared egg extract for 1 h at 18°C. The membranes were sedimented twice by centrifugation and resuspension in buffer. The sample was imaged directly in a confocal microscope. (I) As in H, but the sample was subjected to three freeze–thaw cycles before imaging. (J) GFP-SKL (0.4 µM) was incubated with crude extract for 1 h and imaged. (K) As in J, but 0.1% Triton X-100 was added before imaging. Bars, 10 µM.

Peroxisome protein import in Xenopus egg extract. (A) mScarlet-SKL (0.6 µM) was added at time point zero to cleared Xenopus egg extract. Where indicated, 5 or 10 mM ATPγS was added 20 min before substrate. The samples were immediately mounted on PEG-passivated glass coverslips and imaged at the indicated time points, using a spinning-disk confocal microscope. The mean fluorescence of peroxisomes was determined from >10 images using an automated image analysis script. Shown are the combined data of two different experiments (>20 images per time point), each normalized to the final time point of the control. For each time point, the mean and the standard deviation of the mean are given. (B) Cleared extract was incubated with 0.5 µM GFP-SKL for 1 h at 20–23°C and imaged with a spinning-disk confocal microscope. (C) As in B but at the end of the incubation, 0.75 µM GFP-fluorescence quenching nanobodies (Kirchhofer et al., 2010) were added for ∼15 min before imaging. (D) Quantification of the mean peroxisome fluorescence in B and C of two separate import reactions. Shown are the mean and standard deviation of >20 images. (E) As in B, but with GFP lacking the SKL sequence. (F) As in C, but with GFP lacking the SKL sequence. (G) Quantification of the total fluorescence in E and F of two separate import reactions. Shown are the mean and standard deviation of >20 images. (H) mScarlet-SKL (0.6 µM) was incubated with cleared egg extract for 1 h at 18°C. The membranes were sedimented twice by centrifugation and resuspension in buffer. The sample was imaged directly in a confocal microscope. (I) As in H, but the sample was subjected to three freeze–thaw cycles before imaging. (J) GFP-SKL (0.4 µM) was incubated with crude extract for 1 h and imaged. (K) As in J, but 0.1% Triton X-100 was added before imaging. Bars, 10 µM.

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