Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

Uzoma, Ijeoma; Zhu, Heng

Acetylation is a major epigenetic PTM widely known for its role in regulating chromatin state. However, it is suspected to regulate nonnuclear functions as well [29] . In yeast, no non-histone proteins were reported as substrates of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The catalytic enzyme, Esa1, of the essential nucleosome acetyltransferase of the complex, NuA4, is the only essential HAT in yeast [30] , strongly suggesting that it may mediate acetylation of non-histone proteins critical for cell survival. Another intriguing question was whether HATs could regulate activity of cytosolic proteins or even enzymes like protein kinases. To comprehensively discover the non-chromatin substrates of the NuA4 HAT complex in the yeast proteome, Lin et al. developed in vitro acetylation reactions on the yeast proteome microarrays, containing 5800 yeast proteins, using NuA4 and [ 14 C]-acetyl-CoA [23] . Over 90 non-histone proteins were readily acetylated by the NuA4 complex. Although it was expected that the majority of the substrates would be involved in nucleosome assembly and histone binding categories, a significant number of the identified substrates were cytoplasmic proteins and metabolic enzymes [23] . Twenty proteins involved in a variety of cellular functions such as metabolism, transcription, cell cycle progression, RNA processing and stress response were selected for further validation. Standard double-immunoprecipitation techniques were used to validate 13 of the 20 substrates, including phosphoenolpyruvate carboxykinase (Pck1p). To understand the physiological relevance of nonchromatin acetylation, the authors focused on the cytosolic enzyme Pck1p to explore a connection between acetylation and metabolism. Tandem mass spectrometry (MS/MS) identified lysine 19 (K19) and K514 as the acetylation sites of Pck1p and site-directed mutagenesis revealed that acetylation of K514 is critical for its enzymatic activity and promotes exten- Live mammalian cells Cell surface biomarker identification [9] sion of life span in yeast growing under starvation conditions. These findings demonstrate a functional role for non-chromatin acetylation in yeast metabolism and longevity. Based on GO analysis, acetylation may regulate several other cellular processes as well. In a follow up study, Lu et al. investigated the impact of acetylation on another NuA4 substrate, Sip2, a regulatory subunit of the SNF1 kinase complex (yeast AMPK). Based on the MS/MS analysis and site-directed mutagenesis studies, the authors found that Sip2 acetylation enhances its interaction with the catalytic subunit Snf1 and inhibits Snf1's kinase activity [31] . As a result, phosphorylation of one of Snf1's downstream targets, Sch9 (homolog of Akt/ S6K), is decreased, ultimately leading to slower growth but extended replicative life span. Finally, the authors demonstrated that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. These studies are now echoed by recent discoveries of many mitochondrial and cytosolic enzymes as substrates of acetyltransferases in higher eukaryotes via MS-based PTM profiling [32] [33] [34] .