Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- Institute of Resource Ecology, Helmholtz Institute Dresden-Rossendorf, Germany
Figures
Figure 1 with 3 supplements
Gln1 assembles into filaments in energy-depleted yeast cells.
(A) Yeast cells expressing mCherry-tagged Gln1 from the endogenous promoter were washed twice with water and resuspended in synthetic media (left, control) or citrate buffer of pH 6 (right, …
Figure 1—figure supplement 1
GFP-tagged Gln1 predominantly forms punctate structures.
Yeast cells expressing GFP-tagged Gln1 from the endogenous promoter were washed twice with water and resuspended in synthetic media (left, control) or buffer of pH 6 (right, ‘starved’). White lines …
Figure 1—figure supplement 2
Co-expression of untagged Gln1 transforms the localization pattern from punctate to filamentous.
Yeast cells expressing GFP-tagged Gln1 from the endogenous promoter were washed twice with water and resuspended in synthetic media (left, control) or buffer of pH 6 (right, ‘starved’). The cells …
Figure 1—figure supplement 3
Filamentation is not caused by the tag.
Yeast cells expressing tetracystein-tagged Gln1 were incubated over night with FIAsH-EDT2 to label Gln1. The cells were washed twice with water and resuspended in a phosphate–citrate buffer to …
Figure 2 with 7 supplements
Gln1 assembles by a back-to-back stacking mechanism.
(A) The crystal structure of Gln1 as reported by He at al. (2009). The putative assembly interface and mutations introduced in this study are indicated. (B) Chromosomally encoded Gln1 was replaced …
Figure 2—figure supplement 1
Detailed structural view of the decamer–decamer interface.
All critical residues are shown within a range of 10 Å. Residues mutated in this study are highlighted in red. The subunit identifier is given in brackets. The red arrow denotes the disordered N …
Figure 2—figure supplement 2
Detailed structural view of the decamer–decamer interface.
All critical residues are shown within a range of 10 Å. Residues mutated in this study are highlighted in red. The subunit identifier is given in brackets. The red arrow denotes the disordered N …
Figure 2—figure supplement 3
Detailed structural view of the decamer–decamer interface.
All critical residues are shown within a range of 10 Å. Residues mutated in this study are highlighted in red. The subunit identifier is given in brackets. The red arrow denotes the disordered N …
Figure 2—figure supplement 4
Yeast cells expressing mCherry-tagged R23E Gln1 were subjected to staining with Thioflavin T.
Only the control filaments (formed by the yeast prion Rnq1) were stainable by ThT, suggesting that the formation of cross-β structure is not required for Gln1 filamentation.
Figure 2—figure supplement 5
Lysates from yeast cells expressing wild-type or R23E Gln1 were subjected to semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE).
Only the control filaments (formed by the yeast prion Rnq1) were resistant to SDS, indicating that Gln1 filamentation does not involve the formation of cross-β structure.
Figure 2—figure supplement 6
6xHis-Gln1(R23E)-mCherry was affinity purified from yeast, subjected to negative staining and investigated by electron microscopy.
Note the presence of filaments with a diameter of ∼120 Å and a repeating unit of ∼100 Å in size. These proportions are consistent with the reported dimensions of Gln1 decamers (He et al., 2009). The …
Figure 2—figure supplement 7
Transmission electron microscopy (TEM) was performed on yeast cells expressing untagged Gln1(R23E).
The red arrow points to filamentous structures in the cytoplasm. Similar structures were absent from control cells expressing untagged Gln1. The scale bar is 200 nm.
Figure 3 with 3 supplements
Self-assembly into filaments is driven by macromolecular crowding.
(A) Equal amounts of 6xHis-tagged wild-type and variant Gln1 purified from bacteria were subjected to dynamic light scattering. Shown is the volume distribution that was derived from the intensity …
Figure 3—figure supplement 1
Gel filtration of wild type and variant 6xHis-tagged Gln1 purified from bacteria.
Fractions were applied onto a nitrocellulose filter by using a dot blot apparatus. Molecular weight markers were thyroglobulin (660 kDa), ferritin (440 kDa), catalase (230kD), bovine serum albumin …
Figure 3—figure supplement 2
Electron microscopy of 6xHis-tagged R23E Gln1 purified from bacteria.
Note the formation of chains, which indicate enzyme stacking by a back-to-back mechanism. The scale bar is 20 nm.
Figure 3—figure supplement 3
6xHis-Gln1-mCherry was affinity purified from yeast and assembled in the presence of a crowder.
The sample was investigated by fluorescence microscopy. Acquired images were deconvolved to increase the signal to noise ratio. The shown image is a maximum intensity projection of 20 individual …
Figure 4 with 2 supplements
A drop in intracellular pH triggers filament formation.
(A) Yeast cells expressing Gln1(R23E)-mCherry were spheroplasted and lysed in phosphate buffers of different pHs. Images were acquired immediately after lysis. The scale bar is 5 μm. Note that the …
Figure 4—figure supplement 1
Equal amounts of 6xHis-tagged wild type and E186K Gln1 purified from bacteria were mixed with an acidic buffer and subjected to dynamic light scattering.
Shown is the volume distribution that was derived from the intensity distribution.
Figure 4—figure supplement 2
6xHis-tagged wild-type Gln1 purified from bacteria was subjected to Far-UV CD at pH 7.4 and 6.
The degree of helicity is essentially unchanged as evident from the almost unaffected 222 nm signal. This indicates that Gln1 retains a near-native structure in conditions that induce assembly into …
Figure 5
Other metabolic enzymes form filaments in a pH-dependent manner.
(A) Yeast cells expressing sfGFP(V206R)-tagged Ura8, Glt1, or Gcn3 were washed twice with water and resuspended in buffers of different pHs to induce starvation. Images were taken 2 hr after onset …
Figure 6 with 4 supplements
Assembled Gln1 is catalytically inactive but becomes active again after disassembly.
(A) Lysates were prepared form wild-type cells exposed to advanced starvation conditions for 3, 6, or 24 hr, and the glutamine synthetase activity was determined as described previously (Mitchell …
Figure 6—figure supplement 1
Gln1 filaments form progressively after exposure of yeast to advanced starvation conditions.
Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate–citrate buffer of pH 6 to induce starvation. Images were acquired 0, 3, 6, and 24 hr after onset of …
Figure 6—figure supplement 2
The R23E growth defect can be rescued by adding glutamine to the growth medium.
Endogenous Gln1 was deleted and substituted with wild-type or variant Gln1 expressed from a plasmid. Cells were grown over night and equal amounts of late log phase cells were spotted onto synthetic …
Figure 6—figure supplement 3
The Y81A mutation does not affect growth.
Endogenous Gln1 was substituted with the indicated wild-type or variant versions expressed from an ADH1 promoter-containing plasmid. Cells were grown over night and equal amounts of late log phase …
Figure 6—figure supplement 4
Filament dissolution is impaired in Y81A mutants.
Cells expressing WT or Y81A Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation. After 4 hr of starvation, nutrients were resupplied …
Figure 7
Mechanism of filament formation by Gln1 and its potential role in starvation survival.
(A) Acidification of the yeast cytosol promotes survival and recovery from starvation. Yeast cells were exposed to advanced starvation conditions using buffers with a pH of 6, 7 or 8 for 3 days. …
Videos
Video 1
Gln1 forms filaments in starved yeast. Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation (time point 0). Filament …
Video 2
Gln1 forms filaments in starved yeast. Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation (time point 0). Filament …
Video 3
Gln1 filaments dissolve upon glucose addition to starved cells. Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation …
Video 4
Gln1 filaments dissolve upon glucose addition to starved cells. Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation …
Video 5
Filamentous Gln1 can be reactivated upon entry into the cell cycle. Cells expressing Gln1-mCherry from a GAL-inducible promoter were washed twice with water and starved in a phosphate-citrate buffer …
Additional files
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Supplementary file 1
(A) Plasmids used in this study. (B) Antibodies used in this study. (C) Yeast strains used in this study.
- https://doi.org/10.7554/eLife.02409.034
