Figure 4. Jen1 transits through the TGN during its endocytosis and requires Rod1 for exit from the TGN to the vacuole.
(A) Jen1 co-localizes with the TGN marker, Sec7-mCh, during its trafficking to the vacuole in wild-type cells. WT cells expressing Jen1-GFP and either Vps17-mCh (ySL1168), a marker of the early endosomal compartment, or Sec7-mCh (ySL1165) were grown on lactate medium. Of note, little or no co-localization was observed between Sec7-mCh and Vps17-GFP (Figure 4—figure supplement 1). The Sec7-mCh-expressing cells were labeled with CMAC and were co-injected with the Vps17-mCh-expressing cells into the microfluidics device in lactate medium, before glucose was added. Cells have been cropped to show only one Vps17-mCh expressing cell (left) or one Sec7-mCh expressing cell (right). The uncropped picture is displayed in Figure 4—figure supplement 2, and the full video in Video 6. Co-localization events between Jen1-GFP and either Vps17-mCh or Sec7-mCh are indicated with white arrows or pink arrows, respectively. Scale bar = 2.5 µm. Of note, the co-localization of another transporter, Dip5, with Sec7-mCh after its endocytosis was also observed (see Figure 4—figure supplement 3). (B) Jen1 also co-localizes with the TGN marker, Sec7-mCh, after internalization in the rod1Δ mutant. rod1Δ cells expressing Jen1-GFP and either Vps17-mCh (ySL1177), an endosomal marker, or Sec7-mCh (ySL1176) were grown on lactate medium. As in panel A, the Sec7-mCh-expressing cells were labeled with CMAC and were co-injected with Vps17-mCh-expressing cells into the microfluidics device in lactate medium, before glucose was added. Cells have been cropped to show only one Vps17-mCh expressing cell (left) or one Sec7-mCh expressing cell (right). The uncropped picture is displayed in Figure 4—figure supplement 4, and the full video in Video 7. Co-localization events between Jen1-GFP and either Vps17-mCh or Sec7-mCh are indicated with white arrows or pink arrows, respectively. Scale bar = 2.5 µm. (C) Quantification of the co-localization of Jen1-GFP puncta with either Vps17-mCh or Sec7-mCh puncta in WT (top) or rod1Δ (bottom) cells (20 cells, n = 3). Jen1 co-localizes successively with Vps17 and Sec7. Furthermore, Jen1 co-localizes more robustly with Sec7-mCh in the rod1Δ mutant. See also Figure 4—figure supplement 5. (D) Schematic showing the place of action of the VFT/GARP complex, Ypt6 and the GGA proteins in endosome-to-Golgi trafficking. (E) Deletions of VPS52 (VFT/GARP complex), YPT6 or genes encoding the Ypt6 GEF complex (RGP1 and RIC1) abolish Jen1 trafficking to the vacuole. Lactate-grown WT (ySL1150), vps52Δ (ySL1369), ypt6Δ (ySL1175), ric1Δ (ySL1630) and rgp1Δ (ySL1631) cells expressing Jen1-GFP were imaged before or after the addition of glucose at the indicated time. At t = 30 min Glc treatment, CMAC staining was used to visualize the localization of the vacuole. Scale bar = 5 µm. (F) Deletions of VPS52 or YPT6 prevent the vacuolar degradation of Jen1-GFP. Crude extracts from lactate-grown WT (ySL1150), vps52Δ (ySL1369) and ypt6Δ (ySL1175) cells expressing Jen1-GFP were prepared at the indicated times before and after glucose addition, and were immunoblotted with anti-GFP antibodies to reveal the full-length Jen1-GFP and its degradation product (free GFP). (G) Deletion of YPT6 abrogates Jen1 co-localization with Sec7-mCh. Lactate-grown WT cells (ySL1165) or ypt6Δ cells (ySL1526) expressing Jen1-GFP and Sec7-mCh were injected in a microfluidics device and imaged before (Lactate) and 15 min after glucose addition. Co-localization between Jen1-GFP and Sec7-mCh is indicated with arrowheads. (H) Deletions of GGA1 and GGA2, encoding redundant Golgi-localized clathrin adaptor proteins, alter Jen1 trafficking to the vacuole. Strains gga1Δgga2Δ (ySL1307) or gga1Δ gga2Δ expressing Gga2-HA (ySL1308), used as a positive control, both expressing Jen1-GFP were grown on lactate medium, and imaged before and after the addition of glucose at the indicated times. The gga1Δ gga2Δ cells also express Sec7-mCherry, which allows evaluating of Jen1-GFP co-localization with the TGN (arrowheads). Scale bar = 5 µm. (I) Quantification of the co-localization of Jen1-GFP puncta with Sec7-mCh puncta in gga1Δgga2Δ (ySL1307) cells or gga1Δ gga2Δ cells expressing Gga2-HA (ySL1619) (20 cells, n = 3) over time after glucose addition. Deletion of the GGA genes leads to a transient accumulation of Jen1 at the TGN.
Figure 4—figure supplement 1. Sec7 and Vps17 localize to distinct compartments.
Figure 4—figure supplement 2. Jen1 traffics through the TGN in the course of its endocytosis in wild-type cells.
Figure 4—figure supplement 3. Jen1-GFP and Sec7-mCh co-localize to the same compartment when observed simultaneously.
Figure 4—figure supplement 4. Jen1 also co-localizes to the TGN in rod1Δ mutant cells.
Figure 4—figure supplement 5. Quantification of Sec7-mCh puncta that are also Jen1-GFP positive in WT and rod1Δ cells.
Figure 4—figure supplement 6. Sec7 and Vps17 localize to distinct compartments in rod1Δ cells.
Figure 4—figure supplement 7. Sec7 and Vps17 localize to distinct compartments in gga1Δgga2Δ cells.
Figure 4—figure supplement 8. Dip5-GFP traffics through the TGN in the course of its endocytosis in WT cells.
Figure 4—figure supplement 9. Deletions of GGA1 and GGA2, encoding redundant Golgi-localized clathrin adaptor proteins, alter Dip5 trafficking to the vacuole after endocytosis.
