Specific auxin and medium combinations alter Saccharomyces cerevisiae growth

TL;DR

This study shows that auxin and medium combinations affect yeast growth, suggesting the need for careful controls in experiments using auxin.

Contribution

The novel finding is that auxin affects yeast growth independently of the degradation system, depending on the medium.

Findings

Natural and synthetic auxin impair yeast growth in minimal but not rich media.

Auxin effects are observed regardless of the auxin-inducible degradation system's presence.

Proper controls are essential when using auxin-inducible degradation methods.

Abstract

Auxin-inducible degradation is an increasingly popular protein-targeting reverse genetics approach. Use of this method has revolutionized the types of questions cell and molecular biologists can answer, however, a growing number of studies point to auxin alone impacting different cellular phenotypes. This study investigated the impact of different medium and auxin combinations on
Saccharomyces cerevisiae
growth. We observed that both natural and synthetic auxin (Indole-3-acetic acid (IAA) and 1-Naphthaleneacetic acid (NAA) respectively) impaired budding yeast growth in nutrient minimal but not nutrient rich media. This finding was true across different yeast strains with or without an intact auxin-inducible degradation system. Ultimately, this study highlights the need for proper controls when using auxin-inducible degradation.

Figures

C](#f1) ). Of note, an essential ( *CDC14* ) and non-essential ( *CDC55* ) gene was chosen for this assay *(Powers & Hall, 2017; Visintin et al., 1998; Yellman & Burke, 2006)* . As expected, auxin-inducible degradation of Cdc14-AID significantly impaired yeast growth as previously observed (Powers & Hall, 2017). Conversely, loss of Cdc55-AID led to a modest reduction of growth in minimal medium with IAA. Cdc55-AID loss on rich medium plus IAA or NAA rich or minimal media with NAA had no impact on yeast growth. However, as with wildtype strains, addition of auxin (IAA or NAA) to minimal medium

Tables

Saccharomyces cerevisiae strains
Strain	Genotype	Source
W303a	MATa ura3-52 trp1Δ2 leu2-3_112 his3-11 ade2-1 can1-100	Horizon Discovery - YSC1058
BY4741	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ	Horizon Discovery - YSC1048
KT1112	MATα ura-52 leu2 his3	Kelly Tatchell (Stuart et al., 1994)
YKA890	W303 MATa Cdc14-V5/AID:KanMX	Mark Hall (Powers & Hall, 2017)
YKA892	W303 MATa Cdc14-V5/AID:KanMX leu2::ADH1p-AtTIR1::LEU2	Mark Hall (Powers & Hall, 2017)
YKA1223	W303 MATa CDC55-V5/AID:NAT	Mark Hall
YKA1229	W303 MATa CDC55-V5/AID:NAT trp1::ADH1p-AtTIR1(F79A):TRP1	Mark Hall

Medium
Reagent	Source	Catalogue Number
Yeast Extract	Fisher Scientific	BP1422
Peptone	Fisher Scientific	BP1420
Dextrose	Fisher Scientific	D16
Synthetic complete amino acid supplement w/o Yeast Nitrogen Base	USBiological	D9515
Yeast Nitrogen Base without Amino Acids	Research Products International Corp	Y20040
1-Naphthaleneacetic acid	Sigma Aldrich	N0640
Indole-3-acetic acid	Gold Biotechnology	I-110-25
Agar, Powder	Fisher Scientific	BP1423

Full text

Description

Protein-targeting reverse genetic approaches are becoming increasingly popular. Auxin-inducible degradation represents one of the most widespread methodologies to quickly and temporally remove proteins-of-interest (POI). This system has three parts. First, the plant F-box TIR1 (from Oryza sativa (osTIR1) or from Arabidopsis thaliana (atTIR1)) is exogenously expressed in non-plant eukaryotic cells. TIR1 is able to form a functional SCF E3 ligase with endogenous proteins ( Figure 1A ). Secondly, POI are tagged with the TIR1 recognition motif, auxin-inducible degron (AID). Finally, the plant hormone auxin is added to the system. This induces TIR1-AID-tagged POI interaction leading to polyubiquitination and proteasomal degradation of the POI (Nishimura et al., 2009). Use of this system is becoming increasingly widespread in diverse fields of cell biology and across model organisms (Brown et al., 2017; Camlin & Evans, 2019; Holland et al., 2012; Kanke et al., 2011; Kreidenweiss et al., 2013; Nishimura et al., 2009; Zhang et al., 2015). As a result, auxin-inducible degradation has led to significant advancements in our understanding of cellular functions. However, like all methodological approaches, this system is not without its drawbacks. A growing number of papers have found that auxin is not as inert to non-plant eukaryotic cells as initially believed. For example, in Drosophila auxin was found to alter gene expression (Fleck et al., 2024). Furthermore, in Saccharomyces cerevisiae , the naturally occurring auxin Indole-3-acetic acid (IAA) has been found to impair cell growth, alter the TORC1 pathway, induce filamentation, and impair mRNA movement (Domeni Zali & Moriel-Carretero, 2023; Nicastro et al., 2021; Prusty et al., 2004).

While there is precedent for auxin impacting S. cerevisiae growth, to date no systematic assessment has been performed. Recently our laboratory noticed a previously unpublished phenomenon, variable *S. cerevisiae * growth with specific auxin and medium combinations. Therefore, we set out to systematically test this phenotype across multiple budding yeast strains to determine if this was a universal impact across multiple yeast strains or if this was specific to certain backgrounds. Experiments compared the impact of different auxin types (IAA or 1-Naphthaleneacetic acid (NAA)) on cell growth in nutrient rich (YPD medium) or minimal (SCD medium) conditions ( Figure 1B ). Initial experiments compared three commonly used wildtype yeast strains W303a, BY4741, and KT1112. Auxin (500 µM IAA or NAA) or their vehicle controls (ethanol or NaOH) had no impact on yeast growth compared to control plates (no auxin or vehicle) in nutrient rich medium. Conversely, both IAA and NAA reduced yeast growth on minimal media plates for all wild type strains tested.

To determine if this phenotype was also observed in S. cerevisiae with a functional auxin-inducible degradation system, a second set of experiments were conducted using yeast strains with AID-tagged endogenous proteins with and without integrated atTIR1 ( Figure 1C ). Of note, an essential ( CDC14 ) and non-essential ( CDC55 ) gene was chosen for this assay (Powers & Hall, 2017; Visintin et al., 1998; Yellman & Burke, 2006) . As expected, auxin-inducible degradation of Cdc14-AID significantly impaired yeast growth as previously observed (Powers & Hall, 2017). Conversely, loss of Cdc55-AID led to a modest reduction of growth in minimal medium with IAA. Cdc55-AID loss on rich medium plus IAA or NAA rich or minimal media with NAA had no impact on yeast growth. However, as with wildtype strains, addition of auxin (IAA or NAA) to minimal medium impaired growth in all cells, whether an essential protein was degraded or not. Taken together these results clearly show that choice of auxin medium combination is extremely important for auxin-inducible degradation experiments. Therefore, care should be taken to ensure adequate control for auxin impact is included when using auxin-inducible degradation.

Recently, a new auxin-inducible degradation system, AID2, has been developed. AID2 uses a modified TIR1 and the bulky auxin analog 5-phenyl-indole-3-acetic acid (5-Ph-IAA) (Yesbolatova et al., 2020). A major advantage of AID2 is auxin concentration can be lowered from 500 µM to 1 µM. And importantly, two 2024 studies observed no obvious growth defects with 1 µM 5-Ph-IAA (Gameiro et al., 2024; Valenti et al., 2024). Ultimately, a shift from the original auxin-inducible degradation system to the AID2 system is likely to minimize the negative impact of auxin on S. cerevisiae.

Methods

S. cerevisiae were grown to stationary phase at 30°C in YPD (yeast, peptone, dextrose) medium. Cells were diluted to an OD600 of 1 with sterile water. Ten-fold serial dilutions were spotted onto agar plates (YPD or SCD (synthetic complete + dextrose)) with 500 µM IAA or NAA, vehicle (ethanol for IAA and NaOH for NAA), or with no additive. Plates were grown at 30°C for 48 h with images taken on a ChemiDoc MP Imaging System (BioRad) at 24 and 48 h.

 

 

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