The CS domain of SHQ1 alone binds to NAP57 only in trans to the SSD. (A) Schematic of linear NAP57 constructs used in the study. NAP57 contains a central catalytic (cat) domain that is excised in the NAPΔcat construct. (B) Schematic of a structure model of NAP57 without its unstructured N- and C-terminal extremities (NAP 31–422). N- and C-terminal parts wrap around each other to form a separate domain (light green) from the catalytic part of the enzyme (dark green). (C) Model of full-length NAP57 that highlights the major X-DC mutation cluster and the unstructured C-terminal tail of the NAPΔcat domain (the short N-terminal extremity is not shown). (D) Schematic of linear SHQ1 constructs used in the study. SHQ1 has two major domains, the CS domain (deep red) and the SHQ1-specific domain (SSD, orange). (E) Schematic of a model of the R2TP complex with the heterohexameric ring of the AAA+ ATPases pontin and reptin, and with PIH1D1 and RPAP3. Note all schematics are roughly drawn to scale to each other. (F) Growth of a yeast strain deleted for shq1 and complemented with the yeast SHQ1 constructs indicated on the left, individually (rows 1–4) or combined (row 5). Dilutions (1:1) were spotted left to right. (G–K) Coomassie blue stained SDS-PAGE of recombinant proteins retained on amylose resin by their maltose binding protein (MBP) tags and input (1/10th). Note in all figures, MBP-tagged proteins are highlighted in bold and the added/bound proteins are indicated in regular print. Also, the MBP-tag is omitted when the migration position of the fusion proteins is marked on the side of the gel. As reported previously, all MBP-NAP57 constructs containing its charged and unstructured C terminus migrate as a doublet (Grozdanov et al. 2009a,b; Walbott et al. 2011). (G) Binding of the CS domain of SHQ1 and its SSD in trans to full-length NAP57. The contrast in the boxed area is enhanced. Migrating positions of molecular weight markers (kDa) are indicated on the right. (H) Addition of SSD to MBP-CS and MBP-MS2 phage coat protein (MCP). (I) Binding of the CS domain of SHQ1 and its SSD in trans to NAP57Δcat. (J) Binding of SHQ1 to MBP-NAP57 after RNase treatment of either protein or (K) the same with the CS domain of SHQ1 and its SSD in trans.
