ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion
- Barcelona Institute of Science and Technology, Spain
- Universitat Pompeu Fabra, Spain
- Medical University of Innsbruck, Austria
- National Research Council of Italy, Italy
- European Molecular Biology Laboratory, Germany
- Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain
Figures
Figure 1
A quantitative assay for Acb1 secretion.
(A) The cell wall is a highly-charged, porous meshwork of glucans, chitin, and mannoproteins. Incubation in high pH buffers loosens the cell wall, thus allowing some non-covalently bound proteins to …
Figure 2
Acb1 secretion requires Grh1 and a subset of ESCRT proteins.
Wild type and deletion strains were grown to mid-logarithmic phase, washed twice, and incubated in 2% potassium acetate for 2.5 hr. Cell wall proteins were extracted from equal numbers cells in 100 …
Figure 3
The involvement of ESCRTs in CUPS formation, stability and disassembly.
(A) Wild type and the indicated deletion strains expressing Grh1-2xGFP were grown to mid-logarithmic phase, washed twice, and incubated in 2% potassium acetate for 30 min and 2.5 hr to assess CUPS …
Figure 4 with 1 supplement
Snf7 localizes transiently to CUPS.
(A) Genomically integrated Snf7-RFP and Grh1-2xGFP were visualized by fluorescence microscopy during growth in mid-logarithmic phase and after incubation in 2% potassium acetate for 2.5 hr. (B) …
Figure 4—figure supplement 1
GFP-Cps1 transport is unaffected in pSnf7-RFP expressing cells.
Wild type cells co-expressing GFP-Cps1 (pRS415) with either empty pRS416 vector or pSnf7-RFP were grown mid-logarithmic phase and visualized by fluorescence microscopy. Scale bar = 2 μm.
Figure 5
Recruitment of Snf7 to membranes for Acb1 secretion and MVB pathway share a similar mechanism.
Snf7-RFP was expressed exogenously from its own promoter in wild type and the indicated ESCRT deletion strains expressing Grh1-2xGFP. Cells were visualized by fluorescence microscopy during growth …
Figure 6
Snf7 recruitment to CUPS and Acb1 secretion are accelerated in vps4Δ cells.
(A) Snf7-RFP was expressed exogenously from its own promoter in wild type and vps4Δ cells expressing Grh1-2xGFP. Cells were starved and immediately visualized by time-lapse confocal microscopy …
Figure 7 with 2 supplements
Ultrastructure of CUPS and the involvement of Snf7 in their stability.
CUPS are revealed as a spheroidal collection of highly curved membranes of average 200 nm diameter (WT starvation 1, 3D Model). (A) Grh1-2xmCherry expressing cells were grown to mid-logarithmic …
Figure 7—figure supplement 1
Remaining 3D models of Grh1-positive membranes from Figure 7A.
https://doi.org/10.7554/eLife.16299.011
Figure 7—figure supplement 2
Examples of CLEM analysis from wild type and snf7Δ cells at 45 min of starvation.
Grh1-positive membranes from 12 tomograms each from wild type and snf7Δ cells of were analyzed.
Figure 8
Immunoelectron microscopy identifies Acb1 in stable CUPS.
Grh1-2xGFP expressing cells were starved for 2.5 hr and processed for immunogold labeling and electron microscopy (see Materials and methods). GFP = 15 nm gold, Acb1 = 10 nm gold. Stage 1 – Grh1 …
Figure 9
A schematic presentation of steps in Acb1 secretion.
Grh1 containing vesicles and tubules assemble into foci that are clearly visible as one to two spots per cell. This collection of membranes are formed and consumed constitutively during growth …
Videos
Video 1
Morphology of CUPS Full tomogram and 3D reconstruction of Grh1-2xmCherry positive membranes corresponding to Figure 7A – WT – starvation 1 – 'CUPS alone'.
https://doi.org/10.7554/eLife.16299.013
Video 2
