Figure 9. Plasma membrane–derived cargo... | Rockefeller University Press

Figure 9.

$Plasma membrane–derived cargo can pass through the SEs. (A) Schematic model of α-factor uptake by yeast cells. Green dots refer to SE, green ring to vacuoles (Vac.), and magenta dots to MVB. (B) Trafficking of α-factor relative to SEs. Selected time points from LLSM image (Video 7) after 3D deconvolution are shown. Cells expressing mGFP-tagged Ivy1 were grown in a synthetic medium, cooled to 4°C to block endocytosis, and treated with fluorescent α-factor for 15 min at 4°C. After mounting, α-factor was tracked by LLSM at 23°C. Indicated time points refer to the time interval after 5 min when cells were shifted to 23°C. (C) 3D track mean fluorescence intensities of Ivy1-mGFP and α-factor from Video 7. Respective Ivy1 and α-factor fluorescence intensities were analyzed by vantage time plots in Imaris, the plots statistics values measured by surpass objects with spots. The analyzed dots were indicated in B by white arrows. The experiment was done three times with similar observations. (D) Working model of the SE function in endolysosomal trafficking. Endocytic transport of a plasma membrane protein (red) bound to cargo (blue) occurs via the EE and MVB toward the vacuole. SEs are shown at the interface between the EE and the Golgi. Rab5 (5, green) and Rab7 (7, black) indicate membrane identity of each compartment. Vps4 (4, pink) is present on MVBs, Ivy1 on SEs and the vacuole, where the two pools of EGOC (a substrate of the AP-3 pathway) and TORC1 are also observed. A fraction of EGOC and TORC1 also resides on MVBs. HOPS promotes fusion between these compartments. For details, see text.$

Plasma membrane–derived cargo can pass through the SEs. (A) Schematic model of α-factor uptake by yeast cells. Green dots refer to SE, green ring to vacuoles (Vac.), and magenta dots to MVB. (B) Trafficking of α-factor relative to SEs. Selected time points from LLSM image (Video 7) after 3D deconvolution are shown. Cells expressing mGFP-tagged Ivy1 were grown in a synthetic medium, cooled to 4°C to block endocytosis, and treated with fluorescent α-factor for 15 min at 4°C. After mounting, α-factor was tracked by LLSM at 23°C. Indicated time points refer to the time interval after 5 min when cells were shifted to 23°C. (C) 3D track mean fluorescence intensities of Ivy1-mGFP and α-factor from Video 7. Respective Ivy1 and α-factor fluorescence intensities were analyzed by vantage time plots in Imaris, the plots statistics values measured by surpass objects with spots. The analyzed dots were indicated in B by white arrows. The experiment was done three times with similar observations. (D) Working model of the SE function in endolysosomal trafficking. Endocytic transport of a plasma membrane protein (red) bound to cargo (blue) occurs via the EE and MVB toward the vacuole. SEs are shown at the interface between the EE and the Golgi. Rab5 (5, green) and Rab7 (7, black) indicate membrane identity of each compartment. Vps4 (4, pink) is present on MVBs, Ivy1 on SEs and the vacuole, where the two pools of EGOC (a substrate of the AP-3 pathway) and TORC1 are also observed. A fraction of EGOC and TORC1 also resides on MVBs. HOPS promotes fusion between these compartments. For details, see text.

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