Figure S1.

Degradation and ubiquitination of unassembled chaperonin subunits, related to Figure 1

(A) Diagram illustrating the mRNA coding for the dual-color CCT reporter construct and expected protein products. GFP-fused CCT subunit and RFP are produced from the same mRNA through ribosomal skipping at a viral P2A sequence. The GFP:RFP fluorescence ratio reports the stability of a test GFP-fusion relative to the internal RFP control.

(B) HEK293T cells were transfected with control or CCT reporter constructs and analyzed by flow cytometry. The control construct does not contain a CCT subunit and only has GFP and RFP. The dotted line indicates the peak from the histogram of this control construct. Note that each of the CCT subunit fusions to GFP is less stable than GFP alone, with CCT4 being among the least stable and CCT8 being among the most stable.

(C) ³⁵S-methionine-labeled CCT subunits with a C-terminal TST were translated in RRL (total IVT) and affinity-purified via TST under native conditions. The affinity-purified products were split into three equal aliquots, incubated with E1, E2 (UBCH5), His-Ub, ATP, and RRL, as indicated. The products of this post-purification ubiquitination reaction were either analyzed directly (Ub reac.) or after His-Ub pull-down under denaturing conditions (His-Ub PD). ³⁵S-methionine-labeled CCT subunits were visualized by autoradiography.