Rapamycin r 5000

Chemicals were obtained from the following sources: Concanamycin A (ConcA) from Santa Cruz Biotechnology (SC-202111C); dimethyl sulfoxide (DMSO; D2650), CCCP (C2920), and doxycycline from Sigma Aldrich (D9891); MG-132 (10012628) from Cayman Chemical; hydrogen peroxide (H₂O₂; H325-500) from Fisher Scientific; and rapamycin (R-5000) from LC Laboratories. Source and catalogue numbers for plasmids, antibodies, other reagents, and yeast strains are described in the appropriate sections below. Final reagent concentrations in all experiments were as follows: ConcA = 500 nM; DMSO = 0.4%; CCCP = 10 μM; doxycycline = 20 μg/ml; MG-132 = 55 μM; H₂O₂ = 1 mM; and rapamycin = 1 μg/ml.

Yeast strains were constructed in BY4742 (MATαhis3-1, leu2-0, met15-0, and ura3-0) by homologous recombination of gene-­targeted, PCR-generated DNAs using the method of Longtine et al. (1998) (link). Mutant strains were either derived from the EUROSCARF KANMX deletion collection (Open Biosystems/Thermo Scientific, Waltham, MA) or produced by replacement of the complete reading frame with the HIS3MX6 or URA3 cassette. Gene deletions were confirmed by PCR amplification of the deleted locus. Cells were grown in standard synthetic complete medium lacking nutrients required to maintain selection for auxotrophic markers and/or plasmids (Sherman et al., 1979 ), unless indicated otherwise. To induce nonselective autophagy, cells were grown to early log phase, concentrated, and resuspended in standard synthetic complete medium containing 0.2 μg/ml rapamycin (R-5000; LC laboratories) for 2–4 h at 30°C. For experiments rescuing autophagosomes-vacuole fusion defects, cells were grown overnight to mid–log phase in synthetic complete medium or in synthetic medium containing 50 mM ETA (Sigma-­Aldrich; E6133). For time-course experiments, mid–log phase cells grown in synthetic medium were directly added ETA to a final concentration of 50 mM and then imaged using 60-min time intervals.

The yeast strains used in this study are listed in Table S1. Gene deletion and tagging were performed using standard PCR-based methods, as described previously (Janke et al., 2004 (link); Knop et al., 1999 (link)), and validated by PCR. Cells were cultured at 30°C in YPD medium (1% yeast extract [Gibco], 2% Bacto peptone [Gibco], 2% glucose) or in a synthetic defined medium comprising 0.17% yeast nitrogen base without amino acids and ammonium sulfate (BD), 0.5% ammonium sulfate, 0.5% casamino acids (BD), 2% glucose, 20 µg/ml adenine sulfate, and 20 µg/ml tryptophan (SD/CA medium). To induce autophagy, cells were treated with rapamycin (R-5000; LC Laboratories) at a concentration of 0.2 µM.