Figure 3.
Drs2 and other transmembrane TGN proteins localize with the aid of AP-1/Ent5.(A) Maturation kinetics of Drs2 compared with Sec7 and AP-1. A strain expressing the TGN marker Sec7-mScarlet, the AP-1 subunit Apl2-GFP, and HaloTag-Drs2 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 3. The upper row shows the complete projections, the second row shows edited projections that include only the cisternae being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Two events are shown. Scale bar, 2 µm. (B) Quantification of tagged Golgi proteins during typical maturation events. Depicted are the normalized fluorescence intensities in arbitrary units for the cisternae tracked in A. (C) Smoothed and averaged traces showing the relative kinetic signatures of Drs2, Sec7, and Apl2. Data were obtained for 17 representative cisternae. (D) Mislocalization of transmembrane TGN proteins in apl4Δ ent5Δ cells after CK-666 treatment. apl4Δ ent5Δ cells expressing Sec7-mScarlet and the indicated HaloTag- or GFP-tagged Golgi protein were grown to mid-log phase and then imaged by confocal microscopy 15 min after mock treatment or treatment with CK-666. Shown are average projected Z-stacks. Individual fluorescence channels are shown in grayscale with merged images on the right. Scale bar, 2 µm. (E) Quantification of the effects of the procedure in D for APL4 ENT5 and apl4Δ ent5Δ cells. For a given cell, the individual slices in a Z-stack were examined in the GFP or HaloTag channel to determine whether the cell contained three or more punctate structures with the shape and size characteristics of Golgi cisternae. Each bar represents an average of two biological replicates in which at least 40 cells were scored per condition. Error bars represent SEM.

Drs2 and other transmembrane TGN proteins localize with the aid of AP-1/Ent5. (A) Maturation kinetics of Drs2 compared with Sec7 and AP-1. A strain expressing the TGN marker Sec7-mScarlet, the AP-1 subunit Apl2-GFP, and HaloTag-Drs2 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 3. The upper row shows the complete projections, the second row shows edited projections that include only the cisternae being tracked, and the subsequent rows show the individual fluorescence channels from the edited projections. Two events are shown. Scale bar, 2 µm. (B) Quantification of tagged Golgi proteins during typical maturation events. Depicted are the normalized fluorescence intensities in arbitrary units for the cisternae tracked in A. (C) Smoothed and averaged traces showing the relative kinetic signatures of Drs2, Sec7, and Apl2. Data were obtained for 17 representative cisternae. (D) Mislocalization of transmembrane TGN proteins in apl4Δ ent5Δ cells after CK-666 treatment. apl4Δ ent5Δ cells expressing Sec7-mScarlet and the indicated HaloTag- or GFP-tagged Golgi protein were grown to mid-log phase and then imaged by confocal microscopy 15 min after mock treatment or treatment with CK-666. Shown are average projected Z-stacks. Individual fluorescence channels are shown in grayscale with merged images on the right. Scale bar, 2 µm. (E) Quantification of the effects of the procedure in D for APL4 ENT5 and apl4Δ ent5Δ cells. For a given cell, the individual slices in a Z-stack were examined in the GFP or HaloTag channel to determine whether the cell contained three or more punctate structures with the shape and size characteristics of Golgi cisternae. Each bar represents an average of two biological replicates in which at least 40 cells were scored per condition. Error bars represent SEM.

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