Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.
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We thank Y. Peleg for plasmid construction, G. Brodsky for graphics, R. Rotkopf for support in statistical analysis, K. Tedrick for technical help with the Y2H experiments, and C. Meisinger and N. Vögtle for help with the MTS assignments. We thank G. Krieger for helpful discussions and technical help. We thank C. Ungermann (University of Osnabrück, Germany) and W.-K. Huh (Seoul National University, Seoul, South Korea) for plasmids. The work in the Schuldiner laboratory was supported by ERC CoG Peroxisystem (646604), SFB 1190 from the DFG, a Mitzutani foundation grant, and a VolksWagen foundation grant (93092). The collaborative work on this manuscript done by the Schuldiner, Pines, Herrmann, and Rapaport laboratories was supported by a DIP grant (P17516). Work at the Rachubinski lab was supported by Foundation Grant FDN-143289 from the Canadian Institutes of Health Research. Work in the Michnick lab was supported by Canadian Institutes of Health Research grant MOP-GMX-152556. Work in the Levy lab was supported by Israel Science Foundation grants 1775/12 and 2179/14. U.W. and D.D. are recipients of the Azrieli student-award grant. M.S. is an Incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics.
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 Conditionally expressed proteins regain metabolic regulation when tagged in a native promoter/regulation manner.
(a) NOP1pr-GFP-GAL2 yeast strain before and after native promoter/regulation GFP swap to a NATIVEpr-GFP tag in synthetic medium with either 2% glucose or 2% galactose. (b) NOP1pr-GFP-PHO5 yeast strain before and after native promoter/regulation GFP swap to a NATIVEpr-GFP tag in synthetic medium with or depleted of phosphate. (c) NOP1pr-GFP-SUC2 yeast strain before and after native promoter/regulation GFP swap to a NATIVEpr-GFP tag in synthetic medium with either 2% or 0.2% glucose. All scale bars are 5µm. Imaging of strains was performed a single time. Images represent entire field.
(a) Histogram of the expression levels of fluorophore-protein fusions of the TEF2pr-mCherry, NATIVEpr-GFP and NOP1pr-GFP libraries. a.u., arbitrary units. (b) Scatter plots showing the correlation between protein abundance of NATIVEpr-GFP tagged strains versus C’ GFP tagged strains12. a.u., arbitrary units. R represents two-sided Spearman correlation test score. Quantitation of abundance was preformed once. Strains with a final abundance score lower than 1, were excluded from the data analysis and Spearman correlation tests.
(a) Two MTS-containing proteins, Tuf1 and Coq10, were N’-tagged with and without a generic MTS in the tagging cassette. (b) Native promoter/regulation swapping of N’ SWAT-tagged Tuf1 restores the native MTS targeting to mitochondria. All scale bars are 5µm. Imaging of strains was performed a single time. Images represent entire field. (c) Mitochondrial proteins tagged with a TEF2pr-mCherry without an MTS (d) MTS and TMD prediction analysis for proteins showing mitochondrial localization with the NOP1pr-GFP, NATIVEpr-GFP, and/or C’ GFP tags. Cx(9)C - cysteine-rich domain26, β-barrel domain27. All scale bars are 5µm. Imaging of strains was performed a single time. Images represent entire field.
(a) N’-tagged Ysa1 under its native promoter has a fraction that co-localizes with the mitochondrial marker Tom20-mCherry. (b) C’-tagged Ysa1 has a fraction that co-localizes with the mitochondrial marker Tom20-mCherry. (c) α-tagged Ysa1 is dually localized between mitochondria (M) and cytosol (C) in subcellular fractionation. Total lysate (T) is shown as control. Hsp60 is probed as a mitochondrial protein. Hxk1 is probed as a cytosolic protein. All scale bars are 5µm. Imaging of strains was performed a single time. Images represent entire field.
The proteins Tam41 (a) and Coq2 (b) and their N-terminally truncated versions (∆MTS) were synthesized in the presence of 35S-methionine in reticulocyte lysate. The 35S-methionine-radiolabeled proteins were then incubated with wild type mitochondria for the indicated times. Non-imported protein was removed by proteinase K (PK) treatment. 10% of the protein used per lane was loaded as control. In the samples labeled with VAO, the membrane potential of mitochondria was dissipated before radiolabeled proteins were added. Positions of precursor (pre) and mature (mat) forms are indicated. The graphs show targetP scores of internal matrix targeting sequence-like regions for each residue in the sequences of Coq2 and Tam4125. The blue dashed line marks the processing site in the proteins for MPP, which removes their N-terminal targeting sequences. The nine green boxes indicate the positions of the transmembrane domains of Coq2.
Supplementary Figure 6 Mitochondrial proteins that are differentially localized depending on medium conditions.
NATIVEpr-GFP tagged mitochondrial proteins suspected to be dually localized were visualized under different medium conditions. Cells were grown in glucose 2% mid-log, glycerol 2% mid-log, galactose 2% mid-log, glucose 0.2% mid-log, or glucose 2% stationary for 4 hours before imaging. All scale bars are 5µm. Imaging of strains was performed a single time. Images represent entire field.
S. cerevisiae HF7c cells expressing Gal4-AD and Gal4-BD protein fusions to Pex17 and Inp1 were tested for the ability of the protein fusions to interact by a yeast two-hybrid growth assay. A minus sign (-) denotes empty vector. A serial dilution series was spotted onto -Leu -Trp medium (left) and -His -Leu -Trp medium (right). Growth on -Leu -Trp medium requires the presence of both AD and BD plasmids in cells and is indicative of cell density. Growth on -His -Leu -Trp medium occurs only in the presence of a protein-protein interaction.
Supplementary Figure 8 Scm4 is an outer-membrane mitochondrial protein with both its N′ and C′ termini facing the cytosol.
(a) TEF2pr-VC-SCM4 with a cytosolic-VN co-localizes with the mitochondrial marker MTS-BFP. (b) Proteinase K (PK) protection assay verifies that Scm4 is an outer membrane mitochondrial protein with both its N’ and C’ termini facing the cytosol. Mitochondria were isolated from cells expressing Scm4 (~21 kDa) HA-tagged at either the N’ (HA-Scm4) or C’ (Scm4-HA). Isolated organelles were treated with the indicated amounts of either PK or trypsin. In some samples the detergent Triton X‐100 was added (+TX-100) or the outer membrane of the organelles was ruptured by swelling (+swelling). Immunodecoration was carried out with antibodies against the HA‐tag, the outer membrane protein Tom70 (70 kDa) that is exposed to the cytosol, or the matrix protein Aco1 (90 kDa). Scale bar is 5µm. Imaging of strains was performed a single time. Images represent entire field.
Supplementary Figure 9 The TEF2pr-VC tag with a cytosolic VN helps to determine the topology for membrane-spanning proteins.
Membrane-spanning proteins that showed a signal with the TEF2pr-VC tag and cytosolic VN can be considered as having their N’ facing the cytosol (“in”). C’ topology from33. Program predictions for number of TMDs and N’ topology. NA – Not Available. Scale bar is 5µm. Imaging of strains was performed a single time. Images represent entire field.
(a-c) Original blots for supplementary figure 3c. b and c are the same blots in different exposure times. Black boxes mark the areas used for the figure. The relevant antibody for the boxes is underlined. (d and e) Original blots for supplementary figures 4a and 4b. (f and g) Original blots for supplementary figure 7b.
Supplementary Figures 1-10
All information on strains in the genome-wide SWAT library
Mitochondrial targeting sequence (MTS) predictions
Check PCR and Anchor Seq analysis
Results of peroxisomal protein interaction screen using DHFR PCA
Results of peroxisomal protein interaction screen using Split Venus PCA
All plasmids used in this study
All primers used in this study
Genes that could not be N-terminally tagged with the SWAT cassettes
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Weill, U., Yofe, I., Sass, E. et al. Genome-wide SWAp-Tag yeast libraries for proteome exploration. Nat Methods 15, 617–622 (2018). https://doi.org/10.1038/s41592-018-0044-9
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