Alanine substitutions and phosphorylation levels measured for the engineered Dam1 complexes used in this study.
| Complex . | Ndc80 interaction regions . | Other phospho-sites . | Incorporation of [γ-32P]ATP . | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Interactions . | A . | B . | C . | (NA) . | Moles phosphoryl group per mole Dam1p . | Moles phosphoryl group per mole Ask1p . | Moles phosphoryl group per mole Spc34p . | ||||
| Dam1c subunit . | Dam1p . | Ask1p . | Spc34p . | Dam1p . | |||||||
| Residues . | S257 . | S265 . | S292 . | S200 . | T199 . | S20 . | S31 . | S311 . | |||
| Wild-type | S | S | S | S | T | S | S | S | 2.6 ± 0.41 | ND | 1.2 ± 0.04 |
| S20A | S | S | S | S | T | A | S | S | 2.1 ± 0.10 | ND | 1.0 ± 0.20 |
| ADam1p | S | S | S | A | A | A | S | S | 2.3 ± 0.51 | None | None |
| BAsk1p | A | A | A | S | A | A | S | S | 0.90 ± 0.05 | 0.7 ± 0.09 | None |
| CSpc34p | A | A | A | A | T | A | S | S | 0.77 ± 0.14 | None | 0.93 ± 0.15 |
| ADam1pBAsk1p | S | S | S | S | A | A | S | S | 2.6 ± 0.87 | ND | None |
| ADam1pCSpc34p | S | S | S | A | T | A | S | S | 1.9 ± 0.56 | None | 0.75 ± 0.03 |
| BAsk1pCSpc34p | A | A | A | S | T | A | S | S | 0.80 ± 0.11 | 0.61 ± 0.10 | 0.92 ± 0.14 |
| 6A | A | A | A | A | A | A | S | S | 0.72 | None | None |
| Complex . | Ndc80 interaction regions . | Other phospho-sites . | Incorporation of [γ-32P]ATP . | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Interactions . | A . | B . | C . | (NA) . | Moles phosphoryl group per mole Dam1p . | Moles phosphoryl group per mole Ask1p . | Moles phosphoryl group per mole Spc34p . | ||||
| Dam1c subunit . | Dam1p . | Ask1p . | Spc34p . | Dam1p . | |||||||
| Residues . | S257 . | S265 . | S292 . | S200 . | T199 . | S20 . | S31 . | S311 . | |||
| Wild-type | S | S | S | S | T | S | S | S | 2.6 ± 0.41 | ND | 1.2 ± 0.04 |
| S20A | S | S | S | S | T | A | S | S | 2.1 ± 0.10 | ND | 1.0 ± 0.20 |
| ADam1p | S | S | S | A | A | A | S | S | 2.3 ± 0.51 | None | None |
| BAsk1p | A | A | A | S | A | A | S | S | 0.90 ± 0.05 | 0.7 ± 0.09 | None |
| CSpc34p | A | A | A | A | T | A | S | S | 0.77 ± 0.14 | None | 0.93 ± 0.15 |
| ADam1pBAsk1p | S | S | S | S | A | A | S | S | 2.6 ± 0.87 | ND | None |
| ADam1pCSpc34p | S | S | S | A | T | A | S | S | 1.9 ± 0.56 | None | 0.75 ± 0.03 |
| BAsk1pCSpc34p | A | A | A | S | T | A | S | S | 0.80 ± 0.11 | 0.61 ± 0.10 | 0.92 ± 0.14 |
| 6A | A | A | A | A | A | A | S | S | 0.72 | None | None |
Alanine substitutions blocked phosphorylation at indicated sites (bold A's), so treatment with Ipl1 kinase and ATP led to phosphorylation specifically at sites that remained unmodified (S or T). Phosphorylation levels on Dam1p, Spc34p, and when possible, Ask1p were quantified as described in Materials and methods. NA, not applicable; ND, not determined.