Premium
Intracellular distribution of human SIRT 7 and mapping of the nuclear/nucleolar localization signal
Author(s) -
Kiran Shashi,
Chatterjee Nirupama,
Singh Sapna,
Kaul Sunil C.,
Wadhwa Renu,
Ramakrishna Gayatri
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12346
Subject(s) - nocodazole , biology , subcellular localization , senescence , cytoplasm , nucleolus , microbiology and biotechnology , nuclear localization sequence , gene isoform , cell , biochemistry , gene , cytoskeleton
Sirtuins belong to a class of NAD ‐dependent deacetylases, and include seven distinct isoforms, of which SIRT 7 is the least studied member. In the present study, the subcellular expression of SIRT 7 in primary fibroblasts undergoing senescence was evaluated by immunocytochemistry and immunoblot assay. Expression of nucleolar SIRT 7 in young fibroblast was very prominent, decreased in pre‐senescent cells, and became undetectable in the senescent cells. Interestingly, we observed previously unreported staining for cytoplasmic SIRT 7 in fibroblasts, and report the existence of a steady‐state level of SIRT 7 in cytoplasm. Selective localization of the high‐molecular‐mass (47.5 kDa) SIRT 7 in the cytoplasmic fraction and the low‐molecular‐mass (45 kDa) SIRT 7 in the nuclear fraction was observed in the immunoblot analysis of various cell types. The specificity of the N‐terminal antibodies for detection of cytoplasmic SIRT 7 was confirmed by RNA i and peptide competition assays. The two forms of SIRT 7 showed reciprocal expression following serum starvation, nocodazole and okadaic acid treatments, and also during senescence. Using a combination of deletion constructs and site‐directed mutagenesis, we defined the role of two distinct SIRT 7 sequences in the N‐terminal region (amino acids 61–76, LQGRSRRREGLKRRQE) and the C‐terminal region (amino acids 392–400, KRTKRKKVT) for nuclear and nucleolar import, respectively. In conclusion, we report for the first time the existence of a cytoplasmic pool of SIRT 7 in addition to its well‐known nucleolar form, identify distinct localization signals for its nuclear/nucleolar targeting, and describe an association between loss of nucleolar SIRT 7 and replicative senescence.