Key Resources Table.
Antibodies used. The list of antibodies against S. cerevisiae mitoribosomal proteins includes double nomenclature to avoid confusion.
Oligonucleotides listed were used for creation of MRP mutant strains. All sequences are in 5′-3′ direction. F, forward; R, reverse.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Aco1 | Gift of Dr. Xin Jie Chen | (Chen et al., 2005) |
| Coa3 | Previously generated by our group | (Fontanesi et al., 2011) |
| Cox1 | Anti-MTCO1 antibody [11D8B7] from Abcam | Cat#ab110270 |
| Cyt b | Gift from Dr. Alexander Tzagoloff | (Dieckmann and Tzagoloff, 1985) |
| Mba1 | Gift from Dr. Martin Ott and Dr. Johannes M. Herrmann | (Ott et al. 2006) |
| Mdm38 | Gift from Dr. Johannes M. Herrmann and Peter Rehling | (Bauerschmitt et al. 2010) |
| Oxa1 | Gift from Dr. Martin Ott and Dr. Johannes M. Herrmann | (Ott et al. 2006) |
| Porin | Anti-VDAC1/Porin antibody [16G9E6BC4] - Mitochondrial Loading Control from Abcam | Cat#ab110326 |
| uL2 (Rml2) | This study | Peptide aa379- 392; CRMKVKDRPRGKDAR |
| uL3 (Mrpl9) | This study | Peptide aa207- 220; QNQDPGRVLPGRKM |
| uL6 (Mrpl6) | This study | Purified recombinant protein |
| uL16 (Mrpl16) | Generated by our group | Peptide aa161-174; CAREAFRKAGTKLPG; (De Silva et al., 2017) |
| uL23 (Mrp20) | Gift from Dr. Martin Ott and Dr. Rosemary Stuart | (Gruschke et al., 2010), (Kaur et al., 2011) |
| uL24 (Mrpl40) | Gift from Dr. Rosemary Stuart | (Kaur et al. 2011) |
| bL28 (Mrpl24) | This study | Peptide aa42- 55; DAKPLHMPKERKKFC |
| uL29 (Mrpl4) | Gift from Dr. Martin Ott | (Gruschke et al., 2010) |
| bL31 (Mrpl36) | Gift from Dr. Martin Ott | (Gruschke et al., 2010) |
| bL32 (Mrpl32) | Gift from Dr. Rosemary Stuart | (Kaur et al., 2011) |
| bL33 (Mrpl39) | Generated by our group | Peptide aa57-70; CVAERKPLDFLRTAK; (De Silva et al., 2017) |
| mL50 (Mrpl13) | Gift from Dr. Alexander Tzagoloff | (Jin et al., 1997) |
| bS1 (Mrp51) | Gift from Dr. Martin Ott | (Gruschke et al., 2010) |
| mS37 (Mrp10) | Gift from Dr. Alexander Tzagoloff | (Jin et al., 1997) |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Peptides used to generate antibodies are listed in the antibodies section. All peptides were generated by Genscript. | ||
| Critical Commercial Assays | ||
| SuperSignal West Femto Maximum Sensitivity Substrate | Thermo Scientifc | Cat#34095 |
| Deposited Data | ||
| Mass spectrometry performed with sucrose fractions from mutant strain mitochondrial extracts | This paper | Figure 3 |
| Experimental Models: Organisms/Strains | ||
| αW303-I0 | (Zambrano et al., 2007) | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, ρ+ I0 |
| αW303 / ρ0 | (Zambrano et al., 2007) | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, ρ0 |
| a/αW303-I0 + VAR1 + RNR1 | This study | Diploid, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ρ+ I0 |
| a/αW303-I0 + VAR1 + YMC2 | This study | Diploid, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-YMC2, ρ+ I0 |
| CB11 | (Myers et al., 1985) | MATa, ade1, ρ0 |
| αW303-I0 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ρ+ I0 |
| αW303-I0 + VAR1 + YMC2 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-YMC2, ρ+ I0 |
| αW303 / ρ0+ VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ρ0 |
| αW303-I0 ΔuL1 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL1∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL2 + VAR1 + YMC2 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-YMC2, ΔuL2∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL3 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL3∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL4 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL4∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL5 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL5∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL6 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL6∷KanMX, ρ + I0 |
| αW303-I0 ΔbL9 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL9∷KanMX, ρ + I0 |
| αW303-I0 ΔuL10 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL10∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL11 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL11∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL12 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL12∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL13 + VAR1 + YMC2 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-YMC2, ΔuL13∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL14 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL14∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL15 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL15∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL17 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL17∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL19 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL19∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL21 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL21∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL22 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL22∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL23 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL23∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL24 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL24∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL27 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL27∷KanMX, ρ+ I0 |
| αW303 / I0 ΔbL28 + VAR1 + YMC2 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-YMC2, ΔbL28∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL29 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL29∷KanMX, ρ+ I0 |
| αW303-I0 ΔuL30 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔuL30∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL31 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL31∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL32 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL32∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL34 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL34∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL35 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL35∷KanMX, ρ+ I0 |
| αW303-I0 ΔbL36 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔbL36∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL38 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL38∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL40 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL40∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL41 + VAR1 + YMC2 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-YMC2, ΔmL41∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL43 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL43∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL44 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL44∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL46 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL46∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL49 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL49∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL50 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL50∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL53 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL53∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL54 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL54∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL57 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL57∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL58 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL58∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL59 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL59∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL60 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL60∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL61 + VAR1+ RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, ΔmL61∷KanMX, ρ+ I0 |
| αW303-I0 Δmhr1 + VAR1 + RNR1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, Δmhr1∷KanMX, ρ+ I0 |
| aW303 + YEplac181 | This study | MATa, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, LEU2∷YEplac181, ρ+ |
| aW303 + YMC2 | This study | MATa, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, LEU2∷YEplac181-YMC2, ρ+ |
| aW303 + RNR1 | This study | MATa, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, LEU2∷YEplac181-RNR1, ρ+ |
| aW303-I0 + YEplac181 | This study | MATa, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, LEU2∷YEplac181, ρ+ I0 |
| aW303-I0 + YMC2 | This study | MATa, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, LEU2∷YEplac181-YMC2, ρ+ I0 |
| aW303-I0 + RNR1 | This study | MATa, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, LEU2∷YEplac181-RNR1, ρ+ I0 |
| HHY294 (Δmba1, Δmdm38, oxa1ΔC) | (Bauerschmitt et al., 2010) | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, oxa1-K332*, Δmba1∷HIS5MX6, Δmdm38∷TRP1 |
| αW303-I0 + VAR1 + RNR1 +YEplac112 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112, ρ+ I0 |
| αW303-I0 ΔmL57 + VAR1 + RNR1+YEplac112 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112, ΔmL57∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL57 + VAR1 + RNR1+YEplac112-MBA1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112-MBA1, ΔmL57∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL57 + VAR1 + RNR1+YEplac112-MDM38 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112-MDM38, ΔmL57∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL57 + VAR1 + RNR1+YEplac112-OXA1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112-OXA1, ΔmL57∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL58 + VAR1 + RNR1+YEplac112 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112, ΔmL58∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL58 + VAR1 + RNR1+YEplac112-MBA1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112-MBA1, ΔmL58∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL58 + VAR1 + RNR1+YEplac112-MDM38 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112-MDM38, ΔmL58∷KanMX, ρ+ I0 |
| αW303-I0 ΔmL58 + VAR1 + RNR1+YEplac112-OXA1 | This study | MATα, ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1, URA3∷pRS316-VAR1, LEU2∷YEplac181-RNR1, TRP1:YEplac112-OXA1, ΔmL58∷KanMX, ρ+ I0 |
| Oligonucleotides | ||
| See Supplemental Table S2 | ||
| Recombinant DNA | ||
| pRS316 (URA3) To overexpress ectopic recoded VAR1 | ATCC | Cat#77145 |
| YEplac181 (LEU2) To overexpress either RNR1 or YMC2 | ATCC | Cat# 87588 |
| YEplac112 (TRP1) To overexpress MBA1, MDM38 or OXA1 | ATCC | Cat# 87590 |
| Software and Algorithms | ||
| Mascot algorithm | (Hirosawa et al., 1993) | Mascot Distiller program http://www.matrixscience.com/home.html |
| R studio software | RStudio Team (2015). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA. | URL http://www.rstudio.com/ |