Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome - PubMed (original) (raw)

Proteasomal preferences for initiation sequences in S. cerevisiae. A, boxplots of corrected median cellular YFP fluorescence (median YFP/RFP values) for cultures expressing fluorescent proteasome substrates with different tails at their C termini (pPGK1 dsRed, pTPI1 UbL-YFP-tail; tail sequences shown in

supplemental Table S1

). Whiskers contain data within 1.5 interquartile range (IQR) of box. The IQR of the data is the difference between the 3rd quartile (75th percentile) and 1st quartile (25th percentile) and thus corresponds to the height of the box. B, boxplots (1.5 IQR whiskers) of YFP/RFP values for proteasome substrates in which the UbL domain was replaced with a DHFR domain (DHFR-YFP-tail) in the E1 temperature-sensitive strain uba1-204 at the permissive (30 °C, gray) and restrictive (37 °C, red) temperatures and after proteasome inhibition (30 °C + 100 μ

m

bortezomib, black). N-deg, YFP substrate with N-end rule degron. C, graph plots for the recovery of YFP/RFP values upon proteasome inhibition against the YFP/RFP values of each proteasome substrate. The recovery was calculated as the ratio of the median YFP/RFP values for cultures grown after addition of 100 μ

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bortezomib and DMSO. For stable YFP proteins, the YFP/RFP value does not change upon proteasome inhibition (recovery ≈1), and for well degraded proteins, the YFP/RFP value recovers as the proteasome is inhibited (recovery >1). D, graph plots median YFP/RFP values for cell cultures expressing proteasome substrates expressed at high levels (pPGK1 dsRed, pTPI1 UbL-YFP-tail) against the median YFP/RFP values for the same protein expressed at moderate levels (pACT1 dsRed, pACT1 UbL-YFP-tail). The correlation coefficient is calculated for a fit to a straight line. E, median YFP/RFP values of cell cultures expressing proteasome substrates with different tails at their C termini at moderate expression levels (pACT1 dsRed, pACT1 UbL-YFP-tail, black circles) in the E1 temperature-sensitive strain uba1-204 at the permissive (30 °C) or the restrictive (37 °C) temperature. A ubiquitination-dependent N-end rule substrate is also shown (Ub-R-KK-YFP-Su9, red circle). The median YFP/RFP values for each construct were calculated from 10,000 cells collected in one flow cytometry run. Data points in panels C, D, and E represent mean values determined from at least three repeat experiments; error bars indicate S.E.