Temperature-dependent regulation of proteins in Aspergillus flavus: whole organism stable isotope labeling by amino acids.
Stable isotope labeling by amino acids in cell culture (SILAC) has been used in many different organisms including yeast, mammalian cells, and Arabidopsis cell culture. We present an adaptation of this method to quickly quantify protein changes in response to environmental stimuli regulating biosynthesis of the carcinogen aflatoxin in the fungus Aspergillus flavus. Changes in relative protein concentrations in response to temperature were quantified and compared to changes in aflatoxin biosynthesis and the transcription of the aflatoxin biosynthetic genes. In a comparison between conducive (28 degrees C) and nonconducive (37 degrees C) temperatures for aflatoxin biosynthesis, 31 proteins were found to be more abundant at 37 degrees C and 18 more abundant at 28 degrees C. The change in expression of the aflatoxin pathway enzymes closely followed the strong repression of both aflatoxin biosynthesis and transcription of the aflatoxin pathway genes observed at 37 degrees C. Transcripts corresponding to the 379 proteins quantified by SILAC were analyzed using microarrays, but their expression did not always correlate well with transcript levels of encoding genes. This is the first reported labeling of a multicellular free-living prototroph using the SILAC procedure to compare (13)C(6)-arginine-labeled samples to (12)C(6)-arginine-labeled samples for quantitative proteomics. The data presented shows the utility of this procedure in quantifying changes in protein expression in response to environmental stimuli.
Department of Plant Pathology, Center for Integrated Fungal Research, North Carolina State University, Raleigh, North Carolina 27695, USA.
D Ryan
DR
Georgianna
Adam M
AM
Hawkridge
David C
DC
Muddiman
Gary A
GA
Payne
D000348:Aflatoxins / Q000096:biosynthesis*; D000596:Amino Acids / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D001231:Aspergillus flavus / Q000378:metabolism*; D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D003470:Culture Media; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D007553:Isotope Labeling; D020411:Oligonucleotide Array Sequence Analysis; D053719:Tandem Mass Spectrometry; D013696:Temperature
D000348:Aflatoxins; D000596:Amino Acids; D002247:Carbon Isotopes; D003470:Culture Media; D005656:Fungal Proteins; D001120:Arginine