Figure 1. Peroxisome targeting of... | Rockefeller University Press

Figure 1.

$Figure 1. Peroxisome targeting of SKL-containing fluorescent proteins in Xenopus egg extracts. (A) Cleared egg extract was incubated with 0.9 µM purified mCherry-SKL for 1 h at 18°C. The formation of bright puncta was visualized with a spinning-disk confocal microscope. (B) As in A, but with mCherry lacking the SKL targeting sequence. (C) As in A, but in the presence of a synthetic peptide (300 µM) with a C-terminal SKL sequence. (D) As in C, but with a peptide containing a scrambled import signal at its C terminus (KLS). (E) Cleared egg extract was incubated with 0.6 µM purified mScarlet-SKL for 5 h at 18°C. The sample was subjected to flotation in a discontinuous sucrose gradient. Fractions were collected and analyzed with a fluorescence microscope. Shown is the bottom fraction containing nonimported substrate. (F) As in E, but for the top fraction, containing peroxisome-associated substrate. (G) Quantification of the number of fluorescent peroxisome foci in the different fractions of the sucrose gradient. Shown are the mean and standard deviation from >40 images of two different experiments. (H) All fractions of the sucrose gradient were analyzed by SDS-PAGE, followed by immunoblotting with antibodies against the peroxisome membrane protein Pex14. Bars, 5 µm.$

Peroxisome targeting of SKL-containing fluorescent proteins in Xenopus egg extracts. (A) Cleared egg extract was incubated with 0.9 µM purified mCherry-SKL for 1 h at 18°C. The formation of bright puncta was visualized with a spinning-disk confocal microscope. (B) As in A, but with mCherry lacking the SKL targeting sequence. (C) As in A, but in the presence of a synthetic peptide (300 µM) with a C-terminal SKL sequence. (D) As in C, but with a peptide containing a scrambled import signal at its C terminus (KLS). (E) Cleared egg extract was incubated with 0.6 µM purified mScarlet-SKL for 5 h at 18°C. The sample was subjected to flotation in a discontinuous sucrose gradient. Fractions were collected and analyzed with a fluorescence microscope. Shown is the bottom fraction containing nonimported substrate. (F) As in E, but for the top fraction, containing peroxisome-associated substrate. (G) Quantification of the number of fluorescent peroxisome foci in the different fractions of the sucrose gradient. Shown are the mean and standard deviation from >40 images of two different experiments. (H) All fractions of the sucrose gradient were analyzed by SDS-PAGE, followed by immunoblotting with antibodies against the peroxisome membrane protein Pex14. Bars, 5 µm.