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. 2015 Dec 28;4:e13232. doi: 10.7554/eLife.13232

Figure 5. Effects of anchoring COPI or COPII on general secretion.

(A) Anchoring COPII inhibits secretion more strongly than anchoring COPI. The yeast strains used here all expressed the mitochondrial anchor OM45-FKBPx4. Where indicated, a strain also expressed either Sec31-FRBx2 to anchor COPII, or Sec21-FRBx2 to anchor COPI. Cells growing at 30°C were either left untreated or treated for 10 min with 1 μg/mL rapamycin (“Rap”), then pulsed for 10 min with 35S amino acids, then chased for 30 min. The culture medium was analyzed by SDS-PAGE and autoradiography to detect secreted proteins that had been labeled during the pulse. Numbers represent the molecular weight in kDa of marker proteins. After anchoring COPI, the arrow marks a band of secreted protein that was only partially diminished, and the arrowhead marks a band of secreted protein that was severely diminished. (B) Efficient anchoring of Sec21 requires two copies of FRB with a mitochondrial anchor but only one copy of FRB with a ribosomal anchor. General secretion was visualized by a pulse-chase procedure as in (A), except that the control strain expressed neither an anchor nor an FRB-tagged protein. Where indicated, a strain expressed either a ribosomal Rpl13A-FKBPx2 anchor or a mitochondrial OM45-FKBPx2 or OM45-FKBPx4 anchor, plus either Sec21-FRB or Sec21-FRBx2.

DOI: http://dx.doi.org/10.7554/eLife.13232.011

Figure 5.

Figure 5—figure supplement 1. Insensitivity of cellular protein synthesis to rapamycin.

Figure 5—figure supplement 1.

The experimental procedure was identical to the one in Figure 5A, except that cellular proteins were analyzed rather than proteins secreted into the culture medium. A control (data not shown) confirmed that as in Figure 5A, rapamycin inhibited general secretion completely when COPII was anchored away, but only partially when COPI was anchored away. Numbers represent the molecular weight in kDa of marker proteins.
DOI: http://dx.doi.org/10.7554/eLife.13232.012
Figure 5—figure supplement 2. Time course of the rapamycin effect on general secretion.

Figure 5—figure supplement 2.

The yeast strain expressed the mitochondrial anchor OM45-FKBPx4, and also expressed Sec21-FRBx2 to anchor COPI. Cells growing at 30°C were either left untreated or treated for the indicated times with 2 μg/mL rapamycin (“Rap”), then pulsed for 10 min with 35S amino acids, then chased for 30 min. The culture medium was analyzed by SDS-PAGE and autoradiography to detect secreted proteins that had been labeled during the pulse. Numbers represent the molecular weight in kDa of marker proteins.
DOI: http://dx.doi.org/10.7554/eLife.13232.013
Figure 5—figure supplement 3. Partial inhibition of secretion after anchoring different COPI subunits.

Figure 5—figure supplement 3.

(A) Growth was monitored with or without rapamycin addition as in Figure 2. The parental strain carried the OM45-FKBPx4 anchor. Where indicated, the strains also carried Sec21-FRBx2 or Ret1-FRBx2 or both. (B) A strain carrying the OM45-FKBPx2 anchor was modified to express Ret1-FRBx2-GFP and Sec21-mCherry. Confocal microscopy revealed that addition of 1 μg/mL rapamycin (“Rap”) for 10 min at 23°C anchored both tagged COPI subunits to mitochondria, which were labeled with mito-TagBFP. Scale bar, 2 μm. (C) The strains in (A) were subjected to pulse-chase analysis to detect general secretion as in Figure 5A.
DOI: http://dx.doi.org/10.7554/eLife.13232.014