Ntag fused proteins are degraded via the N-end Rule pathway.

The N-end Rule pathway requires an adaptor protein ClpS that promotes substrate toward the ClpAP protease complex (A). Native proteins are modified to have N-degrons via the Aat transferase enzyme or other mechanisms. These proteins with N-degrons are similarly facilitated by ClpS to the ClpAP complex for degradation as the Ntag fused target proteins. Another system tags native proteins with C-degrons, tags signaling degradation on the C-terminus. These proteins are preferentially degraded in the ClpXP complex by the SspB adaptor protein but can be degraded in the ClpAP complex. The apparent degradation rate (β) for the Ntag-RFP construct was measured across different knockouts of these degradation pathway members in comparison to the ΔclpP strain (B). The presence of N-end Rule members (ClpA, ClpS, ClpP) was required for degradation as indicated by no detectable degradation rate. The absence of ClpX reduced degradation to zero as well. Deletion of Aat resulted in, perhaps, enhanced degradation, and removal of the SspB adaptor protein reduced degradation but did not eliminate it. Single * designates p < 0.05 and triple (***) designates p < 0.001. Calculations of p-values are between ΔclpP and the given condition. Values are mean ± s.e.m. (n = 4 to 10).