NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

Nevertheless, intracellular localization of endogenous NOA36 might be a more complex issue since not only is it located in the nucleolus; but it is also in the mitochondria [36] . In this sense we found that, besides the NoLS, NOA36 could contain a cytoplasmic retention signal located at the carboxy terminus. This fact can be deduced from the localization of the truncated protein 240-310 fused to eGFP. This 36.9 kDa fusion protein should passively diffuse across the nuclear pore complex and enter the nucleus. However, it does not seem to accumulate in the nucleus and it shows mainly cytoplasmic localization ( Fig. 2A) . Cytoplasmic antibody allows the signals of non-transfected cells (NT) and positive transfected cells (NoLS-scPPX) to be separated. Analysis was performed using ImageJ 1.43u software. Results represent the mean 6 S.D. of the measurements in 150 cells in each condition (n = 3). Asterisk indicates significant differences in comparison with non transfected cells, determined by T-test (P,.002). C) Western blot expression of Flag-polyphosphatase protein in cell extracts of cells transfected with an empty vector (pIRES), or with a vector with the NoLS-scPPX construct (pIRES-NoLS-scPPX). Protein molecular weight markers are shown in the middle of the panel (MW), and their estimated protein sizes (in kDa) are indicated to the right. The expected molecular weight for the polyphosphatase protein (,50 kDa) is indicated with an arrow. Nonspecific bands around 85 and 30 kDa appear for all analyzed samples. A representative experiment is shown (n = 3). doi:10.1371/journal.pone.0059065.g005 retention signals (CRS) or cytoplasmic localization signals (CLS) have been previously reported in several human proteins, like Cycline B2 [53] , the HIV-1 Host Defense Factor APOBEC3G [54] and PTEN [55] . Nevertheless endogenous NOA36 is not a cytosolic, but a mitochondrial protein. However, the characterization of the signal responsible of NOA36 mitcochondrial localization remain elusive in our hands, basically, due to the fact the reporters we have used so far (eGFP, RFP, mCherry and Flag) seem to interfere with the mitochondrial localization of the protein. In the case of the Flag-NOA36 construct, the protein localizes at the nucleoli, although can be also detected in the cytoplasm of the 12.5% of transfected cells in a diffuse or punctuated pattern (Fig. 1D) . On the other hand when the NoLS is removed from NOA36 (construct Flag-34C), the recombinant protein shows a nuclear but not nucleolar localization (Fig. 2C) , which would suggest the presence of an additional nuclear localization signal within this truncated protein. Further investigations will be required to clarify the role of additional intracellular transport signals in NOA36.