KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Chemicals | ||
| Anisomycin | Sigma Chemical | A-9789 |
| BMH-21 | MedChemExpress | HY-12484/CS-3557 |
| Cycloheximide | Sigma-Aldrich | C7698 |
| Rapamycin | Millipore | 553211 |
| Thiolutin | N. Belcher, Pfizer | lot 1325-56-1 |
| Zymolyase 20T | MP Biochemicals | 320921 |
| [5,6-3H]-uridine | Perkin-Elmer | cat # NET367 |
| dimethylsulfoxide (DMSO) | Fisher | D128 |
| glucose | Sigma | G8270 |
| NaCl | Sigma | S7653 |
| EDTA | Sigma | E6511 |
| MOPS | Sigma | M1254 |
| formamide | Sigma-Aldrich | 221198 |
| Agarose | Invitrogen | 15510–027 |
| paraformaldehyde | Sigma-Aldrich | 158127 |
| formaldehyde (37%) | Sigma-Aldrich | 252549 |
| vanadate complex (protect RNA RNase inhibitor) | Sigma | R7397 |
| poly-L-lysine | Sigma-Aldrich | P4707 |
| dextran sulfate | Sigma | S8906 |
| xylene cyanol | Sigma-Aldrich | X4126 |
| bromphenol blue | Sigma-Aldrich | B0126 |
| Software and Algorithms | ||
| SoftWorX deconvolution software | Applied Precision, Inc. | http://www.sussex.ac.uk/gdsc/intranet/pdfs/softWoRx%20user%20manual.pdf |
| Excel (for calculating standard deviations) | Microsoft | N/A |
| Experimental Models: Strains of S. cerevisiae | ||
| S. cerevisiae panel of strains expressing single GFP-tagged proteins MATa His+ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 | Invitrogen/Thermo Fisher | Yeast Deletion and Yeast GFP Collections |
| MATα Sik1-mRFP G418R | W-K. Huh | ATY1513 |
| MATα ss-mRFP-HDEL | this study | ATY3196 |
| MATα Htb2-mRFP | this study | ATY3847 |
| kar1Δ15 MAT α CAN1 RNR1 lys2Δ his3–11,15 ura3–1 trp1–1 leu2–3,112 RAD5 W303 | R. Rothstein W2108–14C | ATY4263 |
| MATα kar1Δ15 mRFP-HDEL | this study | ATY6618 |
| MET3-CDC20 (SAC) | this study | ATY10402 |
| MATα Hmo1-Apple | this study | ATY10567 |
| pan-lacO/rDNA MAT a leu2–3,112 trp1–1 can1–100 ura3–1 ade2–1 his3–11,15 ade2:: pAFS144-wtGFP (ADE2) rDNA::pTM-lacO50 (URA3) W303 | T. Kobayashi, TMY3 | ATY10658 |
| pan-lacO/rDNA Sik1-mCherry | As for ATY10658 Sik1-mCherry (KANr) | ATY10659 |
| MATα SAC Hmo1-Apple pan-lacO | this study | ATY10682 |
| MATa SAC Hmo1-Apple pan-lacO | this study | ATY10688 |
| SAC Hmo1-Apple Utp15-CFP Mak11-GFP | this study | ATY10694 |
| SAC Ytm1-Apple mRFP-[pGFP-HDEL] | this study | ATY10717 |
| SAC Mak11-GFP mRFP-HDEL | this study | ATY10722 |
| MATα top1::HIS top4–2 can1–100 his3–11, 15 leu2–3, 112 trp1–1 ura3–1 W303 | R. Rothstein, W1477–5B | ATY10730 |
| MATa SAC pan-lacO Sik1-mRFP | this study | ATY10747 |
| top1Δ top2–4 pan-lacO Sik1-mCherry | this study | ATY10795 |
| Oligonucleotides | ||
| 5’-CTTGCCCAGTAAAAGCTCTCATGCTCTTGC/3Cy3 | IDT | ITS1 probe |
| 5’-CATTATACCTCAAGCACGCAGAGAAACCTC/3Cy5 | IDT | ITS2 probe |
| 5’-AAAGAAGAAGAAGAAGAAGA AGAAGGATAAGA AGAAGGACAAATCCAACTCTTCTATTGGTGACGGT GCTGGTTTA-3’ | Forward primer | for C-terminal tagging Hmo1 with Apple |
| 5’-TTATTATTATATTTATTTTAGAAAGACAGTAGAGT AATAGTAACGAGTTTGTCCGTCCATCGATGAATTCG AGCTCG-3’ | Reverse primer | |
| Plasmids | ||
| pKar2Frag-mRFP-HDEL (URA3/Yip) | N. Dean | pTi-kmRFP |
| pSS-GFP-HDEL (URA3/CEN) | W. Prinz | PWP1055 |
| pMET3-CDC20 (LEU2/Yip22) | R. Uhlmann | p640 |
| pGFP-Nop1 (YCPLAC33 URA3/CEN) | C. Horigome | pNop1 |
| yoApple HIS3 | K.Thorn/M.Garabedian | Apple tag |