Extended Data Fig. 1: TXNL1 is redox active and co-purifies with human 26S proteasomes.
From: Structural landscape of the degrading 26S proteasome reveals conformation-specific binding of TXNL1
a) Purification of TXNL1-bound and TXNL1-free human 26S proteasomes from HEK293 cells. Top: Western blots showing aliquots from the washing (W1-W4) and elution (E) steps in low-salt or high-salt buffer for HTBH-tagged proteasomes that were immobilized on streptavidin agarose. Bottom left: Coomassie-stained SDS-PAGE gel showing the separation of 1 μg human 26S proteasomes purified by size-exclusion chromatography after previous low salt or high salt washes and compared to specific concentrations of recombinant FLAG-tagged TXNL1 purified from E. coli. Bottom right: Western blot of the SDS-PAGE samples on the left, showing TXNL1 levels that co-purified with low-salt or high-salt washed proteasomes in comparison to recombinant His-FLAG-tagged TXNL1. Low-salt washed proteasomes contain sub-stoichiometric amounts of TXNL1, whereas TXNL1 levels for high-salt washed proteasomes are almost undetectable. b) Left: Elution profile for the size-exclusion chromatography (SD75 16/600) of recombinant human TXNL1 that was expressed in E. coli and Ni-NTA affinity purified using its His-(TEV)-FLAG tag. Right: Coomassie-stained SDS-PAGE gel with aliquots from individual stages of recombinant TXNL1 purification. c) Redox activity of recombinant TXNL1 (15 μM) measured by the increase in turbidity (absorbance at 600 nm) that results from the reduction and consequent aggregation of insulin (30 μM). Activities are compared to different concentrations of DTT (top) and TXNL1 mutants that contained only the N-terminal catalytic TRX domain, the C-terminal PITH domain, or the C34S mutation in the catalytic CXXC motif (bottom). d) Degradation of Eos-titinV15P-tail (5 μM) substrate by human 26S proteasome (200 nM) in the absence or presence of excess TXNL1 (15 μM), monitored by the loss of Eos fluorescence. e) FAM fluorescence detection (top) and Coomassie staining (bottom) of the SDS-PAGE gel with samples from the degradation of N-terminally FAM-labeled and ubiquitinated FAMEos-titinV15P-tail substrate (2.5 μM) by human 26S proteasomes (200 nM) in the absence and presence of excess TXNL1 (15 μM). The right 2 lanes show a negative control with non-ubiquitinated substrate (no E1, E2, E3 enzymes).
