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

Ivan S De Melo,Felix A Ruiz,Concepción Iglesias,Antonio Campos-Caro,David Moreno-Sanchez,Maria D Jimenez-Nuñez,Jorge Bolívar,Bruce W Banfield

doi:10.1371/journal.pone.0059065

Ivan S De Melo, Felix A Ruiz + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0059065

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Abstract

NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species.

Highlights

The nucleolus is a dynamic structure that disassembles and reforms during each cell cycle around the rRNA gene clusters [1]
We have previously described the pro-apoptotic role of NOA36 in the mitochondrial apoptotic pathway, the role played by this protein in the nucleolus has not yet been elucidated
Some efforts have been made in order to characterize and classify NoLs [8], the signal that gives rise to this translocalization into the nucleolus is difficult to predict and is sometimes contained within or overlapped by the nuclear localization signal [12]

Summary

Introduction

The nucleolus is a dynamic structure that disassembles and reforms during each cell cycle around the rRNA gene clusters [1]. Nucleolar localization of proteins is mediated typically by functional interaction with nucleolar core components, such as ribosomal DNA, RNA and proteins, yet in most of the cases depends on nucleolar localization sequences (NoLSs) [10]. These kinds of signals have been found in cellular and in viral nucleolar proteins [11]. NoLSs and NLSs have very similar amino acid compositions (a high prevalence of basic residues in both cases) and in some proteins the same region can both target proteins across the nuclear envelope and cause proteins to accumulate in the nucleolus (for example, UTP20 is reported to contain overlapping NLS and NoLS near its C-terminus) [12]. Many nucleolar proteins may accumulate in a steady-state compartment mediated by NoLS (i.e. a sequence or domain) which might interact with local high-affinity binding sites [14]

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