JCS078519 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. No enrichment of Can1−GFP at membrane areas marked by Pil1−mCherry after Pkh1/2 depletion. Surface-view images of pkh1Δpkh2-depletion cells expressing Can1−GFP and Pil1−mCherry before (left panel) and after (right panel) depletion of Pkh2 are shown. Whereas there was a clear enrichment of Can1−GFP in the MCC patches marked by Pil1−mCherry before the depletion, there was no enrichment of Can1−GFP colocalizing with Pil1−mCherry structures after the depletion (see supplementary material Movie 3). Scale bar: 2 µm.

• Supplemental Figure S2 -

  Fig. S2. Endocytosis rate of Can1−GFP triggered by arginine addition is independent of its MCC enrichment. The progress of Can1−GFP internalization after its endocytosis had been triggered by addition of arginine to a concentration of 5 mM was monitored in wild-type cells (left panel) and pil1Δ cells (right panel). Two images are shown for each strain at each time point. Scale bar: 5 µm.

• Supplemental Figure S3 -

  Fig. S3. Endocytosis rate of Can1−GFP triggered by cycloheximide addition is independent of its MCC enrichment. (A) The ratio of mean Can1−GFP fluorescence intensity between the plasma membrane and the cell interior (mean ± s.d., n≥12) is shown for induced wild-type cells (λ), induced pil1Δ cells (σ), non-induced wild-type cells (υ), and non-induced pil1Δ cells (ν). The times relate to the time of cycloheximide addition (=0 minutes). Cycloheximide was added to a concentration of 50 µg/ml to cell cultures at a cell density of OD₆₀₀=0.4. (B) Representative Can1−GFP images of wild-type and pil1Δ cells before and 230 minutes after Can1 endocytosis had been induced by addition of cycloheximide. In contrast to the induction by addition of arginine (compare Fig. 5A and supplementary material Fig. S2), Can1−GFP accumulated in inner cell structures, probably endosomes, which are different from the vacuole. Scale bar: 5 µm.

• Supplemental Table S1 -

  Table S1. Strains used in this study.

• Movie 1 -

  Movie 1. Live cell movie of Abp1−GFP (green) and Pil1−mCherry (red) in a wild-type cell. The focus was set to the bottom surface of the cell.

• Movie 2 -

  Movie 2. Live cell movies of Pil1−mCherry and Sla1−GFP in cells depleted of Pkh1/2. Both cells were imaged after 12 hours incubation with 25 µg/ml Doxycyclin at 30°C. Note the different time intervals between the frames in the two panels. Left panel shows Sla1−GFP in green and Pil1−mCherry in red. Right panel shows the Pil1−mCherry signal.

• Movie 3 -

  Movie 3. Live cell movies of Can1−GFP and Pil1−mCherry in cells depleted of Pkh1/2. Top row shows a cell expressing Can1−GFP and Pil1−mCherry with motile Pil1−mCherry patches after depletion. The Can1−GFP signal is on the left, the Pil1−mCherry signal in the middle, and the overlay on the right (Can1−GFP in green, Pil1−mCherry in red). Bottom row shows a cell expressing Can1−GFP and Pil1−mCherry with a stable net-like Pil1−mCherry structure after depletion. The Can1−GFP signal is on the left, the Pil1−mCherry signal in the middle and the overlay on the right (Can1−GFP in green, Pil1−mCherry in red).

• Movie 4 -

  Movie 4. Live cell movies of Sec3−GFP and Pil1−mCherry and Exo70−GFP and Pil1−mCherry in wild-type cells. The focus was set to the bottom surface of the cells. Note the different time intervals between the frames in the two panels. Left panel shows a cell expressing Sec3−GFP (green) and Pil1−mCherry (red). Right panel shows a cell expressing Exo70−GFP (green) and Pil1−mCherry (red).

• Movie 5 -

  Movie 5. Can1−GFP fluorescence recovery after photobleaching in wild-type cells. The times stated in the movie frames refer to the time of the bleach=0 seconds. Note the different time intervals between the frames in the two panels. The arrows indicate the site of photobleaching. Can1 endocytosis was not induced in the cells used for the FRAP experiments. Left panel shows the Can1−GFP signal photobleached in the MCP area. Right panel shows the Can1−GFP signal photobleached in an MCC patch.

• Movie 6 -

  Movie 6. Tat2−GFP fluorescence recovery after photobleaching in a wild-type cell. The Tat2−GFP signal was photobleached in a MCC patch area. Tat2 endocytosis was not induced. The times stated in the movie frames refer to the time of the bleach=0 seconds. The arrow indicates the site of photobleaching.

• Movie 7 -

  Movie 7. Live cell movie of Tat2−GFP and Lsp1−mCherry in a wild-type cell to which tryptophan is added. Tat2−GFP is shown on the left, Lsp1−mCherry in the middle and the overlay (Tat2−GFP in green, Lsp1−mCherry in red) is on the right. Tryptophan was added to a concentration of 0.35 mM. The times stated in the movie frames refer to the time of tryptophan addition=0 seconds.