Figure 6.
A separate class of transmembrane TGN proteins recycle via PVE compartments.(A) Maturation kinetics of Drs2 compared with Vps10. A strain expressing HaloTag-Drs2 and the vacuolar hydrolase receptor Vps10-GFP 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 part 1 of Video 6. 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 Drs2 and Vps10. Data were obtained for 15 representative cisternae. (D) Maturation kinetics of Drs2 compared with Nhx1. The experiment was performed as in A, except with a strain expressing GFP-Drs2 and the Na+/H+ exchanger Nhx1-HaloTag. Shown are average projected Z-stacks at the indicated time points from part 2 of Video 6. Scale bar, 2 µm. (E) Quantification of tagged Golgi proteins during a typical maturation event. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in D. (F) Smoothed and averaged traces showing the relative kinetic signatures of Drs2 and Nhx1. Data were obtained for eight representative cisternae. (G) Dual localization of Vps10 and Nhx1 before and after treatment with CK-666. apl4Δ ent5Δ strains expressing the TGN marker Sec7-mScarlet, the PVE compartment marker Vps8-HaloTag, and either Vps10-GFP or Nhx1-GFP were grown to mid-log phase, labeled with JFX dye, and imaged by 4D 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. Arrowheads mark examples of Sec7-labeled TGN structures that contain Vps10 or Nhx1, and arrows mark examples of Vps8-labeled PVE compartments that contain Vps10 or Nhx1. Scale bar, 2 µm. At the right, the same apl4Δ ent5Δ strains were treated with CK-666 and manually scored for the presence of three or more punctate spots in the GFP channel, as in Fig. 3 E. Error bars represent SEM.

A separate class of transmembrane TGN proteins recycle via PVE compartments. (A) Maturation kinetics of Drs2 compared with Vps10. A strain expressing HaloTag-Drs2 and the vacuolar hydrolase receptor Vps10-GFP 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 part 1 of Video 6. 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 Drs2 and Vps10. Data were obtained for 15 representative cisternae. (D) Maturation kinetics of Drs2 compared with Nhx1. The experiment was performed as in A, except with a strain expressing GFP-Drs2 and the Na+/H+ exchanger Nhx1-HaloTag. Shown are average projected Z-stacks at the indicated time points from part 2 of Video 6. Scale bar, 2 µm. (E) Quantification of tagged Golgi proteins during a typical maturation event. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in D. (F) Smoothed and averaged traces showing the relative kinetic signatures of Drs2 and Nhx1. Data were obtained for eight representative cisternae. (G) Dual localization of Vps10 and Nhx1 before and after treatment with CK-666. apl4Δ ent5Δ strains expressing the TGN marker Sec7-mScarlet, the PVE compartment marker Vps8-HaloTag, and either Vps10-GFP or Nhx1-GFP were grown to mid-log phase, labeled with JFX dye, and imaged by 4D 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. Arrowheads mark examples of Sec7-labeled TGN structures that contain Vps10 or Nhx1, and arrows mark examples of Vps8-labeled PVE compartments that contain Vps10 or Nhx1. Scale bar, 2 µm. At the right, the same apl4Δ ent5Δ strains were treated with CK-666 and manually scored for the presence of three or more punctate spots in the GFP channel, as in Fig. 3 E. Error bars represent SEM.

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