Dipeptides

The yeast strains used in this study are listed in Additional file 4: Table S1. Rich complete medium (YES) and synthetic minimal medium (EMM2) were used for cell culture. These media and other general yeast methods have been described previously (Forsburg and Rhind 2006 (link)). Ammonium chloride (the nitrogen source in EMM2) was replaced with sodium glutamate (0.38% w/v) to evaluate the sensitivity of yeast to anisomycin, hygromycin B, and terbinafine. Dipeptides were purchased from Sigma-Aldrich Japan (Tokyo, Japan), Bachem (Bubendorf, Switzerland), and Kokusan Chemical Co. Ltd. (Tokyo, Japan), and used at 0.2 mM (to support cell growth) or 5 mM (to inhibit proteolysis). Hi-Nute HK soy peptides (provided by Ms. Kitagawa, Fuji Oil, Osaka, Japan) were used at a concentration of 0.1% (w/v).
To monitor proteolysis via the Arg/N-end rule pathway, the GFP-tagged model substrates, Xaa^Nd-GFP and X-Rec8c-GFP, were used as described previously (Fujiwara et al. 2013 (link); Kitamura and Fujiwara 2013 (link)). To express these proteins from the nmt promoter, the cells were grown in thiamine-free EMM2 for at least 18 h at 28°C. To inhibit degradation via the Arg/N-end rule pathway, the cells were treated with Dipeptides for 3–5 h.

General chemicals and reagents were purchased from Fisher Acros (Hampton, NH, USA). Isothiocyanobenzyl-NTA was purchased from Biomass (Rockville, MD, USA). Tentagel S NH₂ resin was purchased from RAPP polymer (Tuebingen, Germany). Dynabeads^TM MyOne^TM Streptavidin C1, Streptavidin-alkaline phosphatase (SAAP), and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) were obtained from Thermofisher (Hampton, NH, USA). Amino acids, coupling reagents, and biotin were purchased from GL-Biochem. BSA (Shanghai, China), lysozyme, and dipeptides were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ni Sepharose 6 FastFlow beads were obtained from GE Healthcare (Chicago, IL, USA).
MALDI-TOF-MS and MALDI-TOF-MS/MS analysis were performed by a Bruker Micro-flex MALDI-TOF mass spectrometer (Billerica, MA, USA) and a Bruker Ultra-flextreme MALDI-TOF/TOF mass spectrometer. The HPLC analysis and HPLC purification were performed using Agilent 1260 Infinity (Agilent, Santa Clara, CA, USA). Positive bead (blue beads) screening was performed with a Nikon SMZ 745T microscope (Nikon, Tokyo, Japan).