:100; Santa-Cruz Biotechnology), anti-3-Nitrotyrosine (1:100; Millipore), and anti-CD11b (1:100; Hybridoma Bank, Iowa City, IA). The secondary antibodies incorporated biotinylated secondary antibodies (1:200; Vector Laboratories, Burlingame, CA) and FITC-conjugated avidin (1:200; Vector Laboratories) or Rhodamine-conjugated avidin (1:200; Invitrogen). Statistical analysis Information were presented as mean S.D. Statistical analyses were performed employing ANOVA with Bonferroni’s post hoc test. A p-value 0.05 was viewed as as statistically important. Acknowledgments This study was supported by the National Fundamental Analysis Plan of China (Project 973) Grant 2011CB504606 (to LZ) and by the NIH grants EY018659, EY012231, EY019309, and P20RR024215 (to JXM). Author Disclosure Statement No conflicts in financial interests.
Technological Innovation and ResourcesAuthor’s Choice2014 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is accessible on line at http://www.mcponline.orgConvergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-treated Yeast Cells*SVytautas Iesmantavicius, Brian T. Weinert, and Chunaram ChoudharyThe target of rapamycin (TOR) kinase senses the availability of nutrients and coordinates cellular growth and proliferation with nutrient abundance. Inhibition of TOR mimics nutrient starvation and results in the reorganization of lots of cellular processes, including autophagy, protein translation, and vesicle trafficking. TOR regulates cellular physiology by modulating phosphorylation and ubiquitylation signaling networks; nonetheless, the worldwide scope of such regulation will not be completely recognized. Here, we applied a massspectrometry-based proteomics method for the parallel quantification of ubiquitylation, phosphorylation, and proteome changes in rapamycin-treated yeast cells. Our data constitute a detailed proteomic evaluation of rapamycintreated yeast with 3590 proteins, 8961 phosphorylation web-sites, and 2299 di-Gly modified lysines (putative ubiquitylation internet sites) quantified. The phosphoproteome was extensively modulated by rapamycin therapy, with a lot more than 900 up-regulated web-sites 1 hour right after rapamycin treatment. Dynamically regulated phosphoproteins had been involved in diverse cellular processes, prominently like transcription, membrane organization, vesicle-mediated transport, and autophagy. A number of hundred ubiquitylation web pages were improved following rapamycin therapy, and about half as lots of decreased in abundance. We located that proteome, phosphorylation, and ubiquitylation modifications converged on the Rsp5-ubiquitin ligase, Rsp5 adaptor proteins, and Rsp5 targets. Putative Rsp5 targets have been biased for enhanced ubiquitylation, suggesting activation of Rsp5 by rapamycin.Ac4ManNAz Rsp5 adaptor proteins, which recruit target proteins for Rsp5-dependent ubiquitylation, were biased for increased phosphorylation.Fmoc-L-Trp(Boc)-OH Moreover, we located that permeases and transporters, that are generally ubiquitylated by Rsp5, were biased for reduced ubiquitylation and reduced protein abundance.PMID:23892746 The convergence of various proteome-level changes around the Rsp5 technique indicates a crucial role of this pathway in theFrom the Novo Nordisk Foundation Center for Protein Analysis, Faculty of Overall health and Health-related Sciences, University of Copenhagen, Blegdamsvej three, 2200 Copenhagen, Denmark Author’s Choice–Final version full access. Received November 1, 2013, and in revised form, June 23, 2014 Published, MCP Papers in Press, June 24, 2014, DOI ten.1074/ mcp.O113.0.