Figure 1.
AP-1 and Ent5 operate at the TGN and are responsible for the persistent TGN localization of internalized FM 4–64.(A) Maturation kinetics of AP-1 and Ent5 compared with Sec7. A strain expressing the TGN marker Sec7-mScarlet, the AP-1 subunit Apl2-GFP, and the clathrin adaptor Ent5-HaloTag was grown to mid-log phase, labeled with JFX dye, and imaged by 4D confocal microscopy. Shown are average projected Z-stacks at the indicated time points from Video 1. The upper row shows the complete projections, the second row shows edited projections that include only the cisterna being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Scale bar, 2 µm. (B) Quantification of tagged Golgi proteins during a typical maturation event. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in A. (C) Smoothed and averaged traces showing the relative kinetic signatures of Sec7, Apl2, and Ent5. Data were obtained for 18 representative cisternae. Lines show mean values, and shaded areas show 95% confidence intervals. (D) Comparison of internalized FM 4–64 in APL4 ENT5 and apl4Δ ent5Δ strains. Cells expressing Sec7-GFP were grown to mid–log phase and incubated with FM 4-64FX (a fixable version of FM 4–64) during a 3-min pulse, followed by a chase with the quencher SCAS. Representative images are shown from the 5- and 10-min time points during the chase. Individual fluorescence channels are shown in grayscale with merged images on the right. Arrowheads mark examples of TGN structures that contain FM 4–64. Scale bar, 2 µm. (E) Quantification of the analysis in D. For the indicated time points, the Sec7-GFP signals were used to create masks to measure the TGN-associated FM 4–64 fluorescence in APL4 ENT5, apl4Δ ENT5, APL4 ent5Δ, and apl4Δ ent5Δ strains. Plotted are the average TGN-associated FM 4–64 signals, normalized to the value in wild-type cells at 5 min. Error bars represent SEM. At least 100 cells of each strain were analyzed per time point.

AP-1 and Ent5 operate at the TGN and are responsible for the persistent TGN localization of internalized FM 4–64. (A) Maturation kinetics of AP-1 and Ent5 compared with Sec7. A strain expressing the TGN marker Sec7-mScarlet, the AP-1 subunit Apl2-GFP, and the clathrin adaptor Ent5-HaloTag was grown to mid-log phase, labeled with JFX dye, and imaged by 4D confocal microscopy. Shown are average projected Z-stacks at the indicated time points from Video 1. The upper row shows the complete projections, the second row shows edited projections that include only the cisterna being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Scale bar, 2 µm. (B) Quantification of tagged Golgi proteins during a typical maturation event. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in A. (C) Smoothed and averaged traces showing the relative kinetic signatures of Sec7, Apl2, and Ent5. Data were obtained for 18 representative cisternae. Lines show mean values, and shaded areas show 95% confidence intervals. (D) Comparison of internalized FM 4–64 in APL4 ENT5 and apl4Δ ent5Δ strains. Cells expressing Sec7-GFP were grown to mid–log phase and incubated with FM 4-64FX (a fixable version of FM 4–64) during a 3-min pulse, followed by a chase with the quencher SCAS. Representative images are shown from the 5- and 10-min time points during the chase. Individual fluorescence channels are shown in grayscale with merged images on the right. Arrowheads mark examples of TGN structures that contain FM 4–64. Scale bar, 2 µm. (E) Quantification of the analysis in D. For the indicated time points, the Sec7-GFP signals were used to create masks to measure the TGN-associated FM 4–64 fluorescence in APL4 ENT5, apl4Δ ENT5, APL4 ent5Δ, and apl4Δ ent5Δ strains. Plotted are the average TGN-associated FM 4–64 signals, normalized to the value in wild-type cells at 5 min. Error bars represent SEM. At least 100 cells of each strain were analyzed per time point.

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