Abstract
The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing Schizosaccharomyces pombe AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for S. pombe that uses the TIR1 from Oryza sativa (OsTIR1) not fused to Skp1. Furthermore, we demonstrate that degradation efficiency can be improved by pairing an OsTIR1 auxin-binding site mutant, OsTIR1F74A, with an auxin analogue, 5′adamantyl-IAA (AID2). We provide evidence for the enhanced functionality of the OsTIR1 AID and AID2 systems by application to the essential DNA replication factor Mcm4 and to a non-essential recombination protein, Rad52. Unlike AID, no detectable auxin-independent depletion of AID-tagged proteins was observed using AID2.
Highlights
The auxin-inducible degron (AID) is an effective method for the rapid depletion of target proteins, allowing control of protein expression and the study of protein function in vivo [1]
We have significantly improved the utility of the auxin-inducible degron system in S. pombe and this should enhance the study of gene function in vivo by more effectively reducing the levels of gene product
As auxin response has been shown to be intrinsically linked to cellular transport inhibitor response 1 (TIR1) concentration [10,32,33], the increase in TIR1 expression level is the probable reason for the concomitant increase in AID efficiency
Summary
The auxin-inducible degron (AID) is an effective method for the rapid depletion of target proteins, allowing control of protein expression and the study of protein function in vivo [1]. TIR1 is part of the E3 ubiquitin ligase SCF-TIR1 (Skp/Cullin/F-box) complex that recruits an E2 ubiquitin-conjugating enzyme and polyubiquitinates AUX/IAA proteins, targeting them for degradation by the proteasome. This degradation pathway can be transferred to non-plant organisms by (1) exogenously expressing the auxin receptor F-box protein, TIR1 and (2) the fusion of an AUX/IAA auxin-inducible degron (AID) to the protein of interest. The fission yeast AID system developed by Kanke et al [10] uses a fusion of the A. thaliana TIR1 (AtTIR1) to the F-box-interacting component of the SCF, Skp (Skp1-AtTIR1, system termed ‘i-AID’). The off -AID system has been used to efficiently regulate various other proteins including Mcm10 [12], Cdc20 [13], Cnd and Smc2 [14] and Bqt1 [15]
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