Landscape of nuclear transport receptor cargo specificity

Mackmull, Marie‐Therese; Klaus, Bernd; Heinze, Ivonne; Chokkalingam, Manopriya; Beyer, Andreas; Russell, Robert B.; Ori, Alessandro; Beck, Martin

doi:10.15252/msb.20177608

Cited by 94 publications

(100 citation statements)

References 73 publications

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“…It is therefore possible that these importins may be able to form non-typical heterodimers which mediate the import of SIRT2. We also observe that SIRT2 nuclear accumulation was not completely blocked by RNAi of the three importins suggesting that SIRT2 may be shuttled into the nucleus by non-importin interactors via a so called "piggy back" mechanism 31 . Indeed, we have also identified a significant number of nuclear proteins which may contribute to this effect.…”

Section: Discussionmentioning

confidence: 79%

Active nuclear import of the deacetylase Sirtuin-2 is controlled by its C-terminus and importins

Eldridge

Pereira

Impens

et al. 2020

Sci Rep

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The NAD-dependent deacetylase Sirtuin-2 (SIRT2) functions in diverse cellular processes including the cell cycle, metabolism, and has important roles in tumorigenesis and bacterial infection. SIRT2 predominantly resides in the cytoplasm but can also function in the nucleus. Consequently, SIRT2 localisation and its interacting partners may greatly impact its function and need to be defined more clearly. In this study we used mass spectrometry to determine the interactomes of SIRT2 in whole cells and in specific cellular fractions; cytoplasm, nucleus and chromatin. Using this approach, we identified novel interacting partners of SIRT2. These included a number of proteins that function in nuclear import. We show that multiple importins interact with and contribute to the basal nuclear shuttling of SIRT2 and that one of these, IPO7 is required for SIRT2 mediated H3K18 deacetylation in response to bacterial infection. Furthermore, we reveal that the unstructured C-terminus of SIRT2 negatively regulates importin-binding and nuclear transport. This study demonstrates that SIRT2 is actively transported into the nucleus via a process regulated by its C-terminus and provides a resource of SIRT2 interacting partners.The Sirtuin family of NAD-dependent deacetylases consists of 7 members (SIRT1-7) which play key protective roles in age-related diseases and act as metabolic-stress response regulators 1 . Despite sharing conserved NAD-binding and catalytic domains Sirtuins have diverse roles across multiple subcellular compartments. SIRT1, 6 and 7 are primarily nuclear proteins; SIRT3, 4 and 5 localise to the mitochondria and SIRT2 is the only Sirtuin which predominantly resides in the cytoplasm 1 . These differences are in large part due to the distinct N-and/ or C-terminal extensions of different Sirtuins 2 . These regions can regulate substrate binding; catalytic activity; contain specialised domains such as nuclear-localisation signals (NLSs), nuclear-export signals (NESs) and mitochondrial-targeting sequences (MTSs) which control subcellular localisation; and serve as platforms for the addition of post-translational modifications (PTMs) which adjust Sirtuin function 3-5 .SIRT2 has been studied primarily for its roles within the cytoplasmic milieu where it was first identified as a tubulin deacetylase 6 . It has since been demonstrated to have regulatory roles during oxidative stress and inflammatory responses via the direct deacetylation of FOXO3 and NF-κB respectively, as well as multiple pathways relating to glucose and lipid metabolism 7 . The functionality of SIRT2 is extended further by its capacity to localise to different cellular compartments including the ER-Golgi intermediate compartment (ERGIC) 8 , mitochondria 9,10 and notably the nucleus and chromatin [11][12][13] . Despite having a predominantly cytoplasmic localisation, SIRT2 is in fact continuously shuttled between the cytosol and the nuclear compartment 11 . During various physiological conditions, such as mitosis 6,14 and Listeria monocytogenes infection ...

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Section: Discussionmentioning

confidence: 79%

Active nuclear import of the deacetylase Sirtuin-2 is controlled by its C-terminus and importins

Eldridge

Pereira

Impens

et al. 2020

Sci Rep

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“…are used (161)(162)(163)(164), the availability of peptide-level data may shed better light on these still poorly understood aspects of PDB-MS.…”

Section: Downloaded Frommentioning

confidence: 99%

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

Samavarchi-Tehrani

Samson

Gingras

2020

Molecular & Cellular Proteomics

154

150

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The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.

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“…Nucleocytoplasmic trafficking mediated by Importin-β carrier proteins is necessary for a variety of cellular functions. The Importin-β family comprises 11 protein-coding genes in humans (KPNB1, IPO4, IPO5, IPO7, IPO8, IPO9, IPO11, IPO13, TNPO1, TNPO2, and TNPO3), and each has its own biological roles and transports unique sets of cargo (Kimura et al, 2017;Mackmull et al, 2017;Kimura and Imamoto, 2014).…”

Section: Introductionmentioning

confidence: 99%

IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers

O'Brien

Steinhart

Lin

et al. 2019

Journal of Cell Biology

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Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand—independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11−/− cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.

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Landscape of nuclear transport receptor cargo specificity

Cited by 94 publications

References 73 publications

Active nuclear import of the deacetylase Sirtuin-2 is controlled by its C-terminus and importins

Active nuclear import of the deacetylase Sirtuin-2 is controlled by its C-terminus and importins

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers

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