An expanded tool kit for the auxin-inducible degron system in budding yeast

Abstract

Fusion of inducible degradation signals, so-called degrons, to cellular proteins is an elegant method of controlling protein levels in vivo. Recently, a degron system relying on the plant hormone auxin has been described for use in yeast and vertebrate cells. We now report the construction of a series of vectors that significantly enhance the versatility of this auxin-inducible degron (AID) system in Saccharomyces cerevisiae. We have minimized the size of the degron and appended a series of additional epitope tags, allowing detection by commercial antibodies or fluorescence microscopy. The vectors are compatible with PCR-based genomic tagging strategies, allow for C- or N-terminal fusion of the degron, and provide a range of selection markers. Application to a series of yeast proteins, including essential replication factors, provides evidence for a general usefulness of the system.

Keywords: auxin; budding yeast; degron; protein degradation; protein stability.

Figure 1

Truncation and tagging of the C-terminal AID degron. (A) Protein structure of IAA17, corresponding to the full-length AID tag, and truncations examined in this study. (B) Protein levels of Rad53 carrying full-length and truncated versions of the AID tag, examined by western blotting of total lysates; PGK served as a loading control. (C) Protein levels of Rad53 carrying the full-length AID tag in comparison with truncated versions extended by a C-terminal 9myc tag. Where indicated, cultures were treated with 1 mm auxin for 1 h before preparation of the lysates. (D) Degradation of Rad53 by means of the AID tag renders cells sensitive to hydroxyurea (HU) and ultraviolet (UV) radiation. Growth inhibition was monitored on YPD plates containing auxin and/or HU or treated with the indicated UV doses as noted

Figure 2

Variation of epitopes for detection of the AID* tag. (A) Protein levels of Rfa1 carrying a C-terminal AID* tag extended by myc, HA, FLAG or GFP, examined by western blotting with the respective antibodies. Where indicated, cultures were treated with 1 mm auxin for 1 h before preparation of the lysates. Note that the anti-myc blot also detects myc-tagged TIR1 (marked *). (B) Growth curves of the strains described in (A), cultured at 30 °C in YPD medium. (C) Degradation time course of Rfa1 carrying the tags described in (A), after treatment with 1 mm auxin. (D) Growth inhibition by degradation of tagged Rfa1, monitored on YPD plates containing the indicated concentrations of auxin

Figure 3

Variation of selection markers for the AID* tag. Protein levels of Rfa1^AID*–9myc carrying the indicated selection markers were monitored in the presence and absence of auxin, as described in Figure 1C; *, myc-tagged TIR1

Figure 4

Construction of N-terminal AID* tags. (A) Schematic representation of different N-terminal AID* constructs. (B) Protein levels of Rfa1 carrying the N-terminal AID* tags under control of the RFA1 or CUP1 promoter were analysed as described in Figure 1C; *, myc-tagged TIR1. Culture medium contained 0.1 mM CuSO₄. (C) Growth inhibition by degradation of N-terminally tagged Rfa1, monitored as in Figure 2D in the presence or absence of 0.1 mM CuSO₄

Figure 5

Effects of auxin on various AID*-tagged proteins. (A, B) Levels of the indicated proteins carrying the C-terminal AID*–9myc tag after incubation of the respective strains in the presence and absence of 1 mm auxin for 1 h. Myc-tagged TIR1 (marked *) was used as a loading control. (C) Growth inhibition by degradation of the tagged proteins, monitored on YPD plates containing 1 mm auxin where indicated. (D) Cell cycle profiles of the strains used in (A), collected at the indicated times after addition of 1 mm auxin; AS, asynchronous culture