Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5
- Institut Jacques Monod, Université Paris-Diderot, CNRS, France
Figures
Figure 1
Dual function of Rod1 in transporter internalization and post-endocytic sorting.
(A) Rod1 is required for the glucose-induced endocytosis of Stl1, the glycerol/proton symporter, from the plasma membrane to the vacuole. WT (ySL1146) and rod1Δ (ySL1153) cells were grown in …
Figure 2 with 1 supplement
Jen1 ubiquitylation is required for its glucose-induced endocytosis.
(A) Schematic of the lysine-to-arginine mutations introduced in the cytosolic loops of Jen1 to generate the Jen1-KR construct. (B) Jen1-KR-GFP is not ubiquitylated in response to glucose. WT cells …
Figure 2—figure supplement 1
Jen1-KR-GFP is a functional protein.
Jen1 transports selenite (McDermott et al., 2010), which is used here as a readout for Jen1 activity. WT and jen1Δ strains carrying either an empty vector (Ø), a plasmid-encoded Jen1-GFP (pSL161) or …
Figure 3
Rod1 is dynamically recruited to the trans-Golgi network when endocytosis is triggered.
(A) Rod1-GFP re-localizes from the cytosol to punctate structures in response to glucose. Lactate-grown cells (ySL542) expressing Rod1-GFP were injected into a microfluidics device in lactate …
Figure 4 with 9 supplements
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 …
Figure 4—figure supplement 1
Sec7 and Vps17 localize to distinct compartments.
WT cells expressing Sec7-GFP and Vps17-mCh (ySL1531) were injected in the microfluidic device and observed every minute during 9 min. Sec7-GFP-positive vesicles never co-localize with …
Figure 4—figure supplement 2
Jen1 traffics through the TGN in the course of its endocytosis in wild-type cells.
Uncropped pictures corresponding to the panel presented in Figure 3A. See corresponding legend for details. Cells expressing Sec7-mCh were marked with an asterisk. Co-localization events between …
Figure 4—figure supplement 3
Jen1-GFP and Sec7-mCh co-localize to the same compartment when observed simultaneously.
WT cells expressing Jen1-GFP and Sec7-mCh (ySL1165) were grown on lactate medium, and were injected in the microfluidics device in lactate medium, before glucose was added. Cells were imaged at the …
Figure 4—figure supplement 4
Jen1 also co-localizes to the TGN in rod1Δ mutant cells.
Uncropped pictures corresponding to the panel presented in Figure 3B. See corresponding legend for details. Cells expressing Sec7-mCh were marked with an asterisk. Co-localization events between …
Figure 4—figure supplement 5
Quantification of Sec7-mCh puncta that are also Jen1-GFP positive in WT and rod1Δ cells.
This quantification was based on the data presented in Figure 4A,B. Jen1 co-localizes more robustly with Sec7-mCh in the rod1Δ mutant.
Figure 4—figure supplement 6
Sec7 and Vps17 localize to distinct compartments in rod1Δ cells.
rod1Δ cells expressing Sec7-GFP and Vps17-mCh (ySL1602) were injected in the microfluidic device and observed every minute during 9 min. Sec7-GFP-positive vesicles never co-localize with …
Figure 4—figure supplement 7
Sec7 and Vps17 localize to distinct compartments in gga1Δgga2Δ cells.
gga1Δgga2Δ cells expressing Vps17-GFP and Sec7-mCh (ySL1615) were injected in the microfluidic device and observed every minute during 9 min. Vps17-GFP-positive vesicles never co-localize with …
Figure 4—figure supplement 8
Dip5-GFP traffics through the TGN in the course of its endocytosis in WT cells.
WT cells expressing Dip5-GFP and Sec7-mCh (ySL956) were grown on aspartate-free medium, and injected into the microfluidics device in the same medium. Aspartic acid was then added to the medium (200 …
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.
Strains gga1Δgga2Δ (ySL1323) and gga1Δgga2Δ expressing Gga2-HA (ySL1322), used as a positive control, both expressing Dip5-GFP genomically tagged at its endogenous locus were grown on aspartate-free …
Figure 5 with 1 supplement
The prolonged presence of glucose is required for the full endocytosis of Jen1.
(A) Jen1 is deubiquitylated after endocytosis. WT (ySL1150), vps52Δ (ySL1369) and vrp1Δ (ySL1610) cells expressing Jen1-GFP were grown in lactate medium and treated with glucose. Crude extracts were …
Figure 5—figure supplement 1
Jen1 accumulates in an ubiquitylated form in the gga1Δ gga2Δ mutant.
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 crude extracts were prepared before …
Figure 6
The control of Jen1 trafficking at the TGN allows the recycling of internalized transporters back to the cell membrane.
(A) Jen1 endocytosis is reversible upon glucose removal. Lactate-grown WT cells expressing Jen1-GFP (ySL1150) were injected into a microfluidics device in lactate medium. Cells were then imaged over …
Figure 7 with 1 supplement
Rod1 promotes Jen1 exit from the secretory pathway to the vacuole.
(A) A galactose-inducible Jen1-GFP is targeted to the plasma membrane in galactose medium. WT cells (ySL1083) expressing a galactose-inducible Jen1-GFP were grown in raffinose medium overnight, and …
Figure 7—figure supplement 1
The sorting of neosynthesized Jen1 to the vacuole in response to glucose does not require targeting to the plasma membrane and endocytosis.
WT (ySL1083), rod1Δ (ySL781) and vrp1Δ (ySL1650) cells both expressing a galactose-inducible Jen1-GFP were grown overnight on raffinose medium. After 15 min of galactose induction, glucose was then …
Figure 8
Working model for the dual function of Rod1 in the regulation of transporter endocytosis and recycling.
Left, in lactate medium, Jen1 is synthesized and targeted to the plasma membrane. Although Rod1 interacts with Rsp5 (Becuwe et al., 2012b), it is inactive (phosphorylated) and cytosolic. Middle, In …
Author response image 1
Videos
Video 1
Rod1 is required for the glucose-induced internalization of the glycerol/proton symporter Stl1.
WT and rod1Δ (CMAC-positive) cells expressing Stl1-GFP were grown in lactate/glycerol medium and simultaneously observed for 20 min after glucose addition. See also Figure 1C.
Video 2
Jen1-GFP is internalized upon glucose treatment even in the absence of Rod1.
WT cells (left) and in rod1Δ cells (right) expressing Jen1-GFP were grown in lactate medium and observed for 45 min after glucose addition. See also Figure 1D.
Video 3
rod1Δ cells display a kinetic delay in Jen1 internalization.
WT and rod1Δ (CMAC positive) cells expressing Jen1-GFP were visualized simultaneously during 20 min after glucose addition (left). Images of the same video were treated in ImageJ using the ‘Fire’ …
Video 4
Rod1-GFP relocalizes from the cytosol to punctate structures in response to glucose.
WT cells expressing Rod1-GFP were grown in lactate medium and visualized for 60 min after glucose addition. See also Figure 3A.
Video 5
Rod1 co-localizes with the trans-Golgi network marker, Sec7-mCherry, in response to glucose.
WT cells expressing both Rod1-GFP and Sec7-mCh were grown during 4 hr in lactate medium and visualized during 45 min after glucose addition. Merge of Rod1-GFP fluorescence (left panel) and Sec7-mCh …
Video 6
Uncropped video corresponding to Figure 4A.
Jen1-GFP co-localizes with the endosomal marker Vps17-mCh and the TGN marker Sec7-mCh during its trafficking to the vacuole in wild-type cells. WT cells expressing either both Jen1-GFP and Sec7-mCh …
Video 7
Uncropped video corresponding to Figure 4B.
Jen1-GFP co-localizes with the endosomal marker Vps17-mCh and the TGN marker Sec7-mCh during its trafficking to the vacuole in rod1Δ cells. rod1Δ cells expressing either both Jen1-GFP and Sec7-mCh …
Video 8
Jen1 recycles back to the plasma membrane via the TGN.
WT cells expressing both Jen1-GFP and Sec7-mCh, and vps52Δ cells expressing only Jen1-GFP were grown 4 hr in lactate medium and observed simultaneously for 10 min of glucose addition and 20 min …
Video 9
Jen1 recycling correlates with the loss of Rod1-localization to the TGN.
WT cells expressing Rod1-GFP were grown for 4 hr in lactate medium and observed during 3 cycles of glucose addition/removal (5-min pulses). See also Figure 6D.
Video 10
Rod1 is required for the glucose-induced retargeting of Jen1 from the secretory pathway to the vacuole.
WT cells (left panel) and rod1Δ cells (right panel) expressing Jen1-GFP under a galactose-inducible promoter were grown in raffinose medium overnight and simultaneously observed for 15 min during …
Additional files
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Supplementary file 1
A table listing yeast strains used in this study is provided in Supplementary file 1.
- https://doi.org/10.7554/eLife.03307.033
