Cell 65, 917C931.e916 [PMC free article] [PubMed] [Google Scholar] 20. chains on their lysine residues. This ubiquitination is usually mediated by a sequential action of the E1-ubiquitin-activating enzymes, which activate the C-terminal Gly residue of ubiquitin and then transfers it to the E2-ubiquitin carrier family. From there it is attached to a lysine residue of the target protein by highly specific members of the E3-ubiquitin ligase family (8). Although initially the autophagy/lysosome system and the proteasome were regarded as two impartial systems within the cell, in recent years it has become increasingly obvious that those systems are interconnected as both can degrade ubiquitinated substrates. Many proteins have been shown to be degraded by both pathways, and an increased autophagic protein degradation in case of proteasome impairment indicates compensatory mechanisms (9). In this study, we analyzed the impact of proteasomal inhibition around the composition of the Tacrolimus monohydrate proteome of Tacrolimus monohydrate lysosomes and autophagosomes. The activity Tacrolimus monohydrate of the proteasome was inhibited by either BTZ or MG132, and the composition of a lysosome-enriched fraction was analyzed by mass spectrometry. Among other proteins this revealed an increased presence of proteasome complexes within the lysosomal compartment where they are degraded. An in-depth investigation to specify the detailed mechanism revealed that inactivated proteasomes are most likely degraded by macroautophagy and not by chaperone-mediated autophagy but do still reach lysosomes when either ATG5 or ATG7 are Rabbit Polyclonal to ZADH1 deficient. This process is usually neither dependent on Toll-interacting protein (TOLLIP); the adaptor protein for proteaphagy in yeast, nor SQSTM1, and is not accompanied by an increased association of any known macroautophagy adaptor protein to inactivated proteasomes. MATERIALS AND METHODS Antibodies The following antibodies were used: anti-lysosome-associated membrane protein (LAMP)-2 and anti-LAMP1 (Hybridoma Lender, University of Iowa, Iowa City, IA, Clones ABL93 and H4B4 and Clone H4A3, respectively), anti-Cathepsin D (Santa Cruz Biotechnology, Dallas, TX), anti-Proteasome 19S S4 (Abcam, Cambridge, United Kingdom), anti-Proteasome 26S S3 (Abcam), anti-Proteasome subunit (PSMA) 7 (Proteintech, Rosemont, IL), anti-Proteasome subunit (PSMB) 5 (Cell Signaling Technologies, Europe, Leiden, Netherlands), anti-PSMD4 (Proteintech), anti-Tubulin (Sigma-Aldrich, St. Louis, MO), anti-GAPDH (Santa Cruz Biotechnology), anti-Actin (Sigma-Aldrich), anti-TOLLIP (Abcam), anti-LC3 (Novus Biologicals, Centennial, CO), anti-SQSTM1 (Abcam), goat anti-rat IgG Alexa488 (Thermo Fisher Scientific, Waltham, MA). Cell Lines and Treatments Human embryonic kidney (HEK) 293 cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany. MEF cells were generated in-house as described previously (10). Both cell lines were cultured in Dulbecco’s altered Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (Thermo Fisher Scientific), 2 mm l-Glutamine (Thermo Fisher Scientific) and 100 U/ml Penicillin-Streptomycin in a humidified incubator at 37 C and 5% CO2. Unless otherwise noted, for inhibition of the proteasomal activity cells were treated with either 25 m MG132 (Merck KGaA, Darmstadt, Germany) or 1 m BTZ (Merck KGaA) for 5 h, control samples were incubated with the respective amount of dimethyl sulfoxide (DMSO). Plasmids and Generation of CRISPR-CAS Knockout Cell Lines Knockout clones for ATG5, ATG7, TOLLIP, and SQSTM1 were basically generated as described previously (11). In brief, HEK293 cells were transfected with TurboFect? according to the manufacturer’s instructions with the respective target site sequence in lenti-gRNA-GFP-PPT and CAS9 cDNA on pRZ-Flag-mCherry2A-CAS9, which were a kind gift of Prof. Veit Hornung, University of Munich, Germany. Transfection efficiency was verified by fluorescence microscopy and cells were individualized to form single colonies. PCR amplification and data evaluation was performed exactly as described (11). pEGFP-LC3 (human) was a gift from Toren Finkel (Addgene plasmid # 24920; http://n2t.net/addgene: 24920; RRID:Addgene_24920, Watertown, United States) (12). Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isolation of Lysosomes with Magnetic Beads All SILAC reagents were obtained from Thermo Fisher Scientific Tacrolimus monohydrate and Eurisotop, Saint-Aubin, France. For lysosomal proteome analysis, HEK293 cells were cultivated for six passages in SILAC-DMEM supplemented with 10% fetal bovine serum made up of either 87.8 mg/ml l-arginine HCl, 181.2 mg/ml l-lysine for light labeling of cells or l-arginine13C615N4 and l-lysine13C615N2 for heavy labeling of cells. The isolation of lysosomal fractions with magnetic beads was performed as described recently (13). Isolation of Autophagosomes SILAC labeled HEK cells were seeded on Poly-l-lysine (Sigma-Aldrich) coated dishes 1 day before transfection with pEGFP-LC3. 48 h post-transfection, cells were treated with either 1 m BTZ or DMSO control for 5 h. Cells were washed with.