Table 1.
Proteins identified by MALDI–TOF–TOF mass spectrometry from five StrepII and FLAG tag tandem affinity purifications of TFAM or SSBP1, from DNA-enriched mitochondrial supernatants of HEK293T cells (see ‘Materials and Methods’ section)
| A: TFAM-Flag-StrepII | B: SSBP1-Flag-StrepII | C: TFAM-Flag-StrepII | D: SSBP1-Flag-StrepII | E: TFAM-Flag-StrepII |
|---|---|---|---|---|
| ATAD3A | ATAD3A | ATAD3A | ATAD3A | ATAD3A |
| SSBP1 | TFAM | SSBP1 | TFAM | SSBP1 |
| TUFM | TUFM | TUFM | DHX30 | TUFM |
| DHX30 | DHX30 | DHX30 | PTCD3 | PTCD3 |
| PTCD3 | β-actin | PTCD3 | PHB2 | β-actin |
| PHB1 | PHB2 | LRP59 | ||
| CKAP4 | β-actin | CRIF1 | ||
| LRP59 | CKAP4 | |||
| CRIF1 | LRP59 | |||
| MRPS5, MRPS7, MRPS9, MRPS10, MRPS11, MRPS14, MRPS16, MRPS17, MRPS22, MRPS23, MRPS24, MRPS27, MRPS29, MRPS31, MRPS35 | MRPS5, MRPS7, MRPS9, MRPS10, MRPS14, MRPS23, MRPS26, MRPS27, MRPS28, MRPS29, MRPS31, MRPS34 | MRPS5, MRPS9, MRPS10, MRPS14, MRPS22, MRPS26, MRPS27, MRPS28, MRPS29, MRPS31 | MRPS9, MRPS18B, MRPS26, MRPS30 | MRPS5, MRPS9, MRPS10, MRPS22, MRPS23, MRPS26, MRPS27, MRPS28, MRPS29, MRPS31, MRPS34 |
| MRPL4a, MRPL22, MRPL23, MRPL35, MRPL38, MRPL39, MRPL41, MRPL43, MRPL49, ICT1 | MRPL4a, MRPL22, MRPL23, MRPL35, MRPL38, MRPL39, MRPL41, MRPL43, MRPL49, ICT1 | MRPL11, MRPL23, MRPL43 | MRPL4, MRPL11, MRPL15, MRPL19, MRPL28, MRPL37, MRPL38, MRPL44 | MRPL3, MRPL4, MRPL4a, MRPL15, MRPL22 |
| Histones: | Histone: | Histones: | Histones: | Histones: |
| H2B, H4F | H2A2 | H2A2, H2B.1, H3, H4 | H2A, H2B, H4 | H2A, H2B, H3.3, H4 |
The list comprises proteins identified in two or more mitochondrial nucleoid preparations. Proteins identified once only, which do not appear in the table were: leucine rich PPR-motif containing protein (LRP130, or LRPPRC), trifunctional enzyme subunit alpha (HADHA), RNA methyltransferase-like 1, Splicing factor-2 associated protein p32 (SF2P32), dihydrolipoamide branch chain transacylase (DBT), serine hydroxymethyl transferase (SHMT), the mitochondrial phosphate carrier (SLC25A3), C4orf14 (NOA1)—see accompanying article, and protein RPL18a of the 60S cytosolic ribosome. The bait protein was identified in all TAPs (Supplementary Table S1). Abbreviations are as follows: ATAD3, ATPase family AAA domain-containing protein 3, DHX30 (putative ATP-dependent RNA helicase), TUFM (mitochondrial translation elongation factor), PHB 1 or 2 (prohibitin), PTCD3 (pentatricopeptide repeat-containing protein 3), LRP59 (leucine-rich protein 59), CRIF1 or Gadd45GIP1 (growth arrest and DNA damage-inducible proteins-interacting protein 1), the peptidyl tRNA hydrolase, ICT1, CKAP4 (cytoskeletal associated protein 4). Further details of the identification of proteins appear in Supplementary Table S1, and the mitochondrial ribosomal proteins (MRPs) identified in the TAPs are collated in Supplementary Table S2, and shown in parallel with those MRPs identified in subsequent analyses. Histones were for the most part degraded when mitochondria were incubated with trypsin prior to lysis (Supplementary Figure S2). HSPA9 was identified in four TAPs but is not listed in the table, as it was also associated with affinity-purified mitochondrially targeted GFP (Supplementary Figure S3-A).