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

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.

SIRT4-Catalyzed Deacetylation of Axin1 Modulates the Wnt/β-Catenin Signaling Pathway

“…Even though several sirtuin proteins are involved in the Wnt signaling pathway, SIRT4 seems to be the only member that responds rapidly to Wnt stimulation and accumulates in the cytosol. Our work supports the report that SIRT4 is present within both the cytosol and nucleus ( 25 , 26 ). However, how SIRT4 translocates from mitochondria to the cytoplasm and interacts with the destruction complex after Wnt stimulation remains elusive.…”

“…Generally, SIRT4 is supposed to be localized in the mitochondria and is involved in regulating metabolism and metabolic homeostasis ( 23 , 24 ). However, it was recently proved that a small amount of SIRT4 presents in both cytosol and nucleus ( 25 , 26 ). Interestingly, our preliminary results showed that SIRT4 could translocate from mitochondria to the cytoplasm after Wnt stimulation.…”

Research Progress of Sirtuin4 in Cancer

“…Importantly, the N-terminally truncated variant SIRT4 (DN28), which cannot translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. Hence, SIRT4 may exert its anti-proliferative role through mitochondrial/ metabolism-dependent and mitochondria-independent functions (30).…”

Viewing teratogens through the lens of nicotinamide adenine dinucleotide (NAD+)

“…Also, NAD+ and SIRT3, a mitochondrial NAD+ dependent sirtuin, have been shown to control microtubule dynamics and reduce susceptibility to antimicrotubule agents such as vinblastine and colchicine (Harkcom et al, 2014). Additionally, SIRT4, another mitochondrial NAD+ dependent sirtuin, acts as a novel centrosomal/ microtubule-associated protein in the regulation of cell cycle progression (Bergmann et al, 2020).…”