Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein.

Laura Bergmann,Iris Fey,Nina Overbeck,Simone Altinoluk-Hambüchen,Andreas S Reichert,Dennis Sohn,Patrick Verhülsdonk,Constanze Wiek,Helmut Hanenberg,Roland P Piekorz,Christoph Bross,Alexander Lang,Kai Stühler,Anja Stefanski,Jürgen Scheller,Mohammad Reza Ahmadian,Reiner U Jänicke,Christian Mielke,Andreas Kefalas

doi:10.3390/cells9091950

Laura Bergmann, Iris Fey + Show 17 more

Open Access

https://doi.org/10.3390/cells9091950

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Abstract

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

Highlights

Mitotic cell division represents a complex and highly regulated process that allows the equal partitioning of duplicated DNA content from a mother cell into two daughter cells
Similar to endogenously expressed SIRT4, C-terminal eGFP fusion proteins of SIRT4 and SIRT4(∆N28), the latter representing an N-terminally (a.a. 1–28) truncated SIRT4 mutant unable to translocate into mitochondria [42,49], were detected at interphase centrosomes of HT1080 fibrosarcoma and HeLa cervix carcinoma cells (Figure S4 and Video S1)
Truncated SIRT4 mutant unable to translocate into mitochondria [42,49], were detected at interphase centrosomes of HT1080 fibrosarcoma and HeLa cervix carcinoma cells

Summary

Introduction

Mitotic cell division represents a complex and highly regulated process that allows the equal partitioning of duplicated DNA content from a mother cell into two daughter cells. The mitotic spindle apparatus is comprised of two centrosomes, one at each spindle pole, astral and spindle microtubules, and microtubule-associated protein (MAP) complexes [1,2,3]. Centrosomes are the main microtubule organizing centers in animal cells comprising a pair of centrioles that are surrounded by pericentriolar material (PCM) components with pericentrin as the major anchoring factor [4]. The dynamic status of microtubules and their (in)stability are critically regulated by post-translational modifications, including (de)acetylation of α-tubulin (in particular lysine 40 [K40]) [6,7]. The sirtuin SIRT2 and histone deacetylase 6 (HDAC6) are known deacetylases that target K40-acetylated α-tubulin in a NAD+ -dependent manner [8], thereby altering microtubule dynamics by decreasing microtubule stability [9,10]

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