Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking. Author Hanna Budayeva, Ileana Cristea Publication Year 2016 Type Journal Article Abstract Human sirtuin 2 (SIRT2) is an NAD-dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. Keywords Humans, Membrane Transport Proteins, Cells, Cultured, Fibroblasts, Endoplasmic Reticulum, Golgi Apparatus, Protein Transport, Gene Knockdown Techniques, Protein Interaction Maps, Sirtuin 2, Protein Interaction Mapping Journal Mol Cell Proteomics Volume 15 Issue 10 Pages 3107-3125 Date Published 2016 Oct ISSN Number 1535-9484 DOI 10.1074/mcp.M116.061333 Alternate Journal Mol Cell Proteomics PMCID PMC5054338 PMID 27503897 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML