Extended Table 1: Proteomic analysis of complexes resolved by blue native electrophoresis.
CRISPR/Cas9 knockout cells stably rescued with HA-tagged COMMD1 (C1) or HA-tagged VPS26C (26C), were used to immunoisolate CCC and Retriever. The complexes were eluted under native conditions (with HA peptide), resolved in blue native gels, and corresponding bands were subjected to proteomics with iBaqq quantification. Average ratios for each protein against VPS35L, CCDC22, CCDC93, COMMD4 and COMMD9 were calculated to estimate molar ratios.
| 720 MW complex | Spectral counts / MW (sample name) | Ratio to VPS35L | Ratio to CCDC22 | Ratio to COMMD9 | |||
|---|---|---|---|---|---|---|---|
|
| |||||||
| MW | Protein | Control | COMMD1 IP | VPS26C IP | |||
|
| |||||||
| 109,563 | VPS35L | 1822 | 1748200 | 1422000 | 1.000 | 0.446 | |
| 33,010 | VPS26C | 0 | 530920 | 457950 | 0.312 | 0.139 | |
| 20,496 | VPS29 | 75264 | 138070 | 144540 | 0.089 | 0.040 | |
| 40,513 | DENND10 | 3720.5 | 792910 | 521280 | 0.185 | ||
| 70,756 | CCDC22 | 2345.7 | 4090000 | 3015300 | 1.000 | ||
| 73,198 | CCDC93 | 0 | 2588900 | 2013300 | 0.648 | ||
| 21,819 | COMMD9 | 0 | 2003200 | 1968900 | 0.559 | 1.000 | |
| 22,966 | COMMD10 | 0 | 2415900 | 1909600 | 0.609 | 1.089 | |
| 21,764 | COMMD4 | 0 | 1150600 | 1127600 | 0.321 | 0.574 | |
| 24,670 | COMMD5 | 0 | 1075200 | 1264900 | 0.329 | 0.589 | |
| 22,151 | COMMD3 | 0 | 1529200 | 1065500 | 0.365 | 0.653 | |
| 21,178 | COMMD1 | 0 | 1899100 | 1641000 | 0.498 | 0.891 | |
| 22,745 | COMMD2 | 0 | 1247500 | 1061900 | 0.325 | 0.581 | |
| 21,090 | COMMD8 | 0 | 751900 | 596320 | 0.190 | 0.339 | |
| 22,540 | COMMD7 | 0 | 1003500 | 700460 | 0.240 | 0.429 | |
| 9,638 | COMMD6 | 0 | 1581400 | 1202800 | 0.392 | 0.701 | |
|
| |||||||
| 480 MW complex | Spectral counts / MW (sample name) | Ratio to VPS35L | Ratio to CCDC22 | Ratio to COMMD4 | |||
|
| |||||||
| MW | Protein | Control | COMMD1 IP | VPS26C IP | |||
|
| |||||||
| 109,563 | VPS35L | 6365.7 | 852460 | 915390 | 1.000 | 0.120 | |
| 33,010 | VPS26C | 0 | 361700 | 336020 | 0.395 | 0.048 | |
| 20,496 | VPS29 | 67894 | 229980 | 392090 | 0.352 | 0.042 | |
| 40,513 | DENND10 | 0 | 1706100 | 660430 | 0.161 | ||
| 70,756 | CCDC22 | 0 | 8984600 | 5699900 | 1.000 | ||
| 73,198 | CCDC93 | 0 | 4502000 | 2461300 | 0.474 | ||
| 21,764 | COMMD4 | 0 | 3421700 | 2212600 | 0.384 | 1.000 | |
| 21,819 | COMMD9 | 0 | 4004700 | 2489000 | 0.442 | 1.153 | |
| 22,151 | COMMD3 | 0 | 4270600 | 2278200 | 0.446 | 1.162 | |
| 24,670 | COMMD5 | 0 | 2685500 | 1786300 | 0.305 | 0.794 | |
| 22,745 | COMMD2 | 0 | 2876700 | 1956600 | 0.329 | 0.858 | |
| 21,178 | COMMD1 | 0 | 4035300 | 2739700 | 0.461 | 1.202 | |
| 22,966 | COMMD10 | 0 | 4248800 | 2980500 | 0.492 | 1.283 | |
| 21,090 | COMMD8 | 0 | 1549600 | 1319200 | 0.195 | 0.509 | |
| 22,540 | COMMD7 | 0 | 2559000 | 1340400 | 0.266 | 0.692 | |
| 9,638 | COMMD6 | 0 | 3698000 | 2411000 | 0.416 | 1.084 | |
|
| |||||||
| 240 MW complex | Spectral counts / MW (sample name) | Ratio to VPS35L | Ratio to CCDC93 | Ratio to COMMD9 | |||
|
| |||||||
| MW | Protein | Control | COMMD1 IP | VPS26C IP | |||
|
| |||||||
| 109,563 | VPS35L | 0 | 2020800 | 1207400 | 1.000 | 0.632 | |
| 33,010 | VPS26C | 0 | 1428200 | 720550 | 0.666 | 0.421 | |
| 20,496 | VPS29 | 90357 | 1113600 | 1333900 | 0.758 | 0.479 | |
| 40,513 | DENND10 | 0 | 690080 | 223770 | 0.179 | ||
| 73,198 | CCDC93 | 0 | 3679300 | 1427000 | 1.000 | ||
| 70,756 | CCDC22 | 0 | 1688300 | 309660 | 0.391 | ||
| 21,819 | COMMD9 | 0 | 2943600 | 1454200 | 0.861 | 1.000 | |
| 24,670 | COMMD5 | 0 | 2450000 | 972230 | 0.670 | 0.778 | |
| 22,966 | COMMD10 | 0 | 3433200 | 1536200 | 0.973 | 1.130 | |
| 21,764 | COMMD4 | 0 | 1215600 | 120040 | 0.262 | 0.304 | |
| 22,540 | COMMD7 | 0 | 1375800 | 592580 | 0.385 | 0.448 | |
| 21,178 | COMMD1 | 0 | 1019100 | 148690 | 0.229 | 0.266 | |
| 9,638 | COMMD6 | 0 | 1086100 | 75972 | 0.228 | 0.264 | |
| 22,151 | COMMD3 | 0 | 782380 | 100480 | 0.173 | 0.201 | |
| 21,090 | COMMD8 | 0 | 810520 | 111830 | 0.181 | 0.210 | |
| 22,745 | COMMD2 | 0 | 739430 | 127670 | 0.170 | 0.197 | |