Figure 7.
Sys1 localizes with the aid of AP-1/Ent5 but has an intermediate kinetic signature.(A) Maturation kinetics of Sys1 compared with Sec7 and Drs2. A strain expressing Sec7-mScarlet, HaloTag-Drs2, and the transmembrane Golgi protein Sys1-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 7. 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 typical maturation events. 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, Sys1, and Drs2. Data were obtained for 10 representative cisternae. (D) Mislocalization of Sys1 in apl4Δ ent5Δ cells after CK-666 treatment. The experiment was performed as in Fig. 3 D except that Sec2-GFP was also visualized. The budded cell from the untreated culture is typical for the population, while the budded cell from the CK-666–treated culture is a striking example that illustrates the population trend. Scale bar, 2 µm. (E) Quantification of the effects in D. The experiment was performed as in Fig. 3 E, except that apl4Δ ENT5 and APL4 ent5Δ strains were also examined. (F) Maturation kinetics of Sys1 compared with Sec7 in an apl4Δ ent5Δ strain. The experiment was performed as in A, except that HaloTag-Drs2 was not imaged because the signal was too weak. Shown are average projected Z-stacks at the indicated time points from part 2 of Video 7. Scale bar, 2 µm. (G) Quantification of tagged Golgi proteins during typical maturation events in an apl4Δ ent5Δ strain. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in F. (H) Smoothed and averaged traces showing the relative kinetic signatures of Sec7 and Sys1 in an apl4Δ ent5Δ strain. Data were obtained for seven representative cisternae. Error bars represent SEM.

Sys1 localizes with the aid of AP-1/Ent5 but has an intermediate kinetic signature. (A) Maturation kinetics of Sys1 compared with Sec7 and Drs2. A strain expressing Sec7-mScarlet, HaloTag-Drs2, and the transmembrane Golgi protein Sys1-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 7. 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 typical maturation events. 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, Sys1, and Drs2. Data were obtained for 10 representative cisternae. (D) Mislocalization of Sys1 in apl4Δ ent5Δ cells after CK-666 treatment. The experiment was performed as in Fig. 3 D except that Sec2-GFP was also visualized. The budded cell from the untreated culture is typical for the population, while the budded cell from the CK-666–treated culture is a striking example that illustrates the population trend. Scale bar, 2 µm. (E) Quantification of the effects in D. The experiment was performed as in Fig. 3 E, except that apl4Δ ENT5 and APL4 ent5Δ strains were also examined. (F) Maturation kinetics of Sys1 compared with Sec7 in an apl4Δ ent5Δ strain. The experiment was performed as in A, except that HaloTag-Drs2 was not imaged because the signal was too weak. Shown are average projected Z-stacks at the indicated time points from part 2 of Video 7. Scale bar, 2 µm. (G) Quantification of tagged Golgi proteins during typical maturation events in an apl4Δ ent5Δ strain. Depicted are the normalized fluorescence intensities in arbitrary units for the cisterna tracked in F. (H) Smoothed and averaged traces showing the relative kinetic signatures of Sec7 and Sys1 in an apl4Δ ent5Δ strain. Data were obtained for seven representative cisternae. Error bars represent SEM.

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