association of calcineurin with the copi protein sec28 and the copii protein sec13 revealed by quantitative proteomics协会的钙调磷酸酶copi蛋白质sec28和copii sec13揭示了定量蛋白质组学.pdf

Association of Calcineurin with the COPI Protein Sec28 and the COPII Protein Sec13 Revealed by Quantitative Proteomics 1 2 1 2 1 Lukasz Kozubowski *, J. Will Thompson , Maria E. Cardenas , M. Arthur Moseley , Joseph Heitman 1 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America, 2 Duke Proteomics Core Facility, Duke University Medical Center, Durham, North Carolina, United States of America Abstract Calcineurin is a calcium-calmodulin-dependent serine/threonine specific protein phosphatase operating in key cellular processes governing responses to extracellular cues. Calcineurin is essential for growth at high temperature and virulence of the human fungal pathogen Cryptococcus neoformans but the underlying mechanism is unknown. We performed a mass spectrometry analysis to identify proteins that associate with the calcineurin A catalytic subunit (Cna1) in C. neoformans cells grown under non-stress and high temperature stress conditions. A novel prioritization strategy for mass spectrometry data from immunoprecipitation experiments identified putative substrates and proteins potentially operating with calcineurin in common pathways. Cna1 co-purified with proteins involved in membrane trafficking including the COPI component Sec28 and the COPII component Sec13. The association of Cna1 with Sec28 and Sec13 was confirmed by co-immunoprecipitation. Cna1 exhibited a dramatic change in subcellular localization during high temperature stress from diffuse cytoplasmic to ER- associated puncta and the mother-bud neck and co-localized with Sec28 and Sec13. Citation: Kozubowski L, Thompson JW, Cardenas ME, Moseley MA, Heitman J (2011) Association o