Ait1 regulates TORC1 signaling and localization in budding yeast
- Department of Molecular and Cellular Biology, University of Arizona, United States
Figures
Figure 1
The target of rapamycin complex I (TORC1) interactome in budding yeast.
Blue circles show the number of background corrected peptide spectral maps (PSMs) from each protein identified in a specific Kog1 or Pib2 immunopurification, while the red to yellow scale shows the …
Figure 2
Ait1 is a putative seven-helical transmembrane (GPCR-like) protein that localizes to the vacuolar membrane.
(A) The predicted topology of Ait1/Ydl180w from Protter 1.0 (Omasits et al., 2014). The two large cytosolic loops in Ait1, both of which are predicted to be intrinsically disordered (C3 and C4), are …
Figure 3 with 1 supplement
The Ait1 interactome.
Blue circles show the number of background corrected peptide spectral maps (PSMs) for the top 25 proteins identified in the GFP-Ait1 immunopurification (based on the average number of PSMs in the …
Figure 3—figure supplement 1
Target of rapamycin complex I (TORC1) interacts with Ait1.
Immunopurification experiments, carried out after treatment with the dithiobis(succinimidyl propionate) (DSP) crosslinker, confirm that Ait1 interacts with TORC1 in vivo. (A) GFP-Ait1 copurifies …
Figure 4
Target of rapamycin complex I (TORC1)-body formation during nitrogen starvation in strains missing key TORC1 interactors.
Each square on the heat map shows the fraction of cells with a Kog1-YFP focus/body at a specific timepoint, calculated by examining the images of >200 cells, per strain, per timepoint. Replicate …
Figure 5 with 1 supplement
Target of rapamycin complex I (TORC1)-body formation in the ait1Δ strain.
(A) Kog1-YFP localization in the wild-type and ait1Δ strains, during log-phase growth in nutrient-rich (SD) medium. The white bar shows 5 μm. (B) Fraction of wild-type and ait1Δ cells that form …
Figure 5—figure supplement 1
Gtr1 and Pib2 localization in the ait1Δ strain.
(Upper panel) Gtr1-YFP and GFP-Pib2 localization in the wild-type and ait1Δ strains, during log-phase growth in nutrient-rich (SD) medium and 1 hr of complete nitrogen starvation (-N). The white bar …
Figure 6 with 1 supplement
Impact of Ait1 on target of rapamycin complex I (TORC1) activity during amino acid starvation.
(B) TORC1 activity during complete amino acid starvation (top), and leucine starvation (bottom), in wild-type, ait1Δ, and GTR1Q65L (Gtr1on) strains, as measured by western blot using an anti …
Figure 6—figure supplement 1
Impact of Ait1 on target of rapamycin complex I (TORC1) activity during nitrogen and amino acid starvation.
(A) TORC1 activity during complete nitrogen starvation in wild-type and ait1Δstrains, as measured by western blot using an anti phospho-Rps6 antibody. Graphs show the ratio of the p-Rps6 signal …
Figure 7
Impact of Ait1 on target of rapamycin complex I (TORC1) activity in strains with mutations in Gtr1/2, or the prion domains in Kog1, during amino acid starvation.
(A) TORC1 activity during complete amino acid starvation in mutant strains with (left column) and without Ait1 (right column), measured using a western blot, as described in Figure 6. (B) Values …
Figure 8 with 3 supplements
Role of the Ait1 C3 loop in target of rapamycin complex I (TORC1) regulation.
(A) BLAST alignment of the SLC38A9 (top) and Ait1 sequences, showing the entire C3 loop (no other sequences in these proteins align). (B, C) TORC1 activity in Ait1 C3 and C4 loop mutants during …
Figure 8—figure supplement 1
Localization of Ait1 and the Ait1 C3 and C4 loop mutants.
Images taken of strains expressing GFP-Ait1, GFP-AitΔC3, GFP-AitΔC4, GFP-Ait1C3v1, GFP-Ait1C3v2, and GFP-Ait1C3v3 show that the mutants fold and are transported to the vacuolar membrane correctly. …
Figure 8—figure supplement 2
Sequence alignments of Ait1 from a variety of yeast species, showing the high degree of conservation at the center of the C3 loop.
Red dots show the sites mutated in Ait1C3v1 and red and yellow dots show the sites mutated in Ait1C3v2.
Figure 8—figure supplement 3
Condition-dependent signaling changes in Ait1C3v2.
(A–C) Target of rapamycin complex I (TORC1) signaling in the ait1c3v2 and wild-type strains, measured during leucine, complete amino acid, and nitrogen starvation, respectively (as described in Figur…
Figure 9 with 1 supplement
Impact of AIt1 C3 and C4 loop mutations on target of rapamycin complex I (TORC1) localization and Gtr1/2 binding.
(A) Each square on the heat map shows the fraction of cells with a Kog1-YFP focus/body at a specific timepoint (as labeled), calculated by averaging the data from three replicate experiments (>100 …
Figure 9—figure supplement 1
Coimmunoprecipitation of GFP-Ait1 and Gtr1-myc.
(A) GFP-Ait1 and GFP-Ait1C3v2 were immunopurified in strains with or without a myc-tag on Gtr1 (as labeled). As a control we also immunopurified Ait1 from a strain carrying Gtr1-myc, but without a …
Figure 10
Evolution of the target of rapamycin complex I (TORC1) circuit in yeast.
Species identified as carrying Ait1 in a BLAST search (p < 0.001 cutoff) are marked with a red circle on a previously constructed map of Rheb and TSC1/2 conservation among the budding yeast, taken …
Additional files
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Supplementary file 1
Complete data for the Kog1 and Pib2 immunopurifications.
The IP-mock tab shows the number of background corrected peptide spectral counts (PSMs) for each protein detected (rows) across the different IPs (columns). The Filterpass tab shows the number of raw PSMs in each IP and mock IP. Proteins with at least twofold higher abundance in the true IP (Kog1-FLAG or GFP-Pib2) versus the mock IP (Kog1-HA or wild-type Pib2), and with at least seven peptide spectral maps in the true IP, were scored as potential interactors.
- https://cdn.elifesciences.org/articles/68773/elife-68773-supp1-v2.xlsx
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Supplementary file 2
Results of a BLASTP search against Ydl180w from S. cerevisiae.
Subspecies/variants were removed from the table for clarity. No other families of yeast have species with significant E values (p < 0.01).
- https://cdn.elifesciences.org/articles/68773/elife-68773-supp2-v2.xlsx
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Supplementary file 3
List of strains used in this study.
- https://cdn.elifesciences.org/articles/68773/elife-68773-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68773/elife-68773-transrepform1-v2.docx
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Source data 1
Labelled raw gel images.
The original labeled gel, from each panel in Figure 3—figure supplement 1, Figure 6, Figure 6—figure supplement 1, Figure 7, Figure 8, Figure 8—figure supplement 3, Figure 9, and Figure 9—figure supplement 1 are included in source data in two separate folders. In each case the gels are numbered as they are shown in the associated figure—from top to bottom. In the case of Figure 6, the two gels on the left are labeled 1 and 2 and the two gels on the right are labeled 3 and 4.
- https://cdn.elifesciences.org/articles/68773/elife-68773-data1-v2.zip
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Source data 2
Labelled raw gel images.
- https://cdn.elifesciences.org/articles/68773/elife-68773-data2-v2.zip
