A Novel Nuclear Localization Signal in the Auxiliary Domain of Apobec-1 Complementation Factor Regulates Nucleocytoplasmic Import and Shuttling

Valerie Blanc,Susan Kennedy,Nicholas O Davidson

doi:10.1074/jbc.m302951200

Abstract

C to U editing of the nuclear apolipoprotein B (apoB) transcript is mediated by a core enzyme containing a catalytic deaminase, apobec-1, and an RNA binding subunit, apobec-1 complementation factor (ACF). ACF expression is predominantly nuclear, including mutant proteins with deletions of a putative nuclear localization signal. We have now identified a novel 41-residue motif (ANS) in the auxiliary domain of ACF that functions as an authentic nuclear localization signal. ANS-green fluorescence protein and ANS-beta-galactosidase chimeras were both expressed exclusively in the nucleus, whereas wild-type chimeras or an ACF deletion mutant lacking the ANS were cytoplasmic. Nuclear accumulation of ACF is transcription-dependent, temperature-sensitive, and reversible, features reminiscent of a shuttling protein. ACF relocates to the cytoplasm after actinomycin D treatment, an effect blocked by the CRM1 inhibitor leptomycin B. Heterokaryon assays confirmed directly that ACF shuttles in vivo. ACF binds to the protein carrier, transportin 2 in vivo, and colocalizes to the nucleus as determined by confocal microscopy. Co-immunoprecipitation experiments revealed that transportin 2 binds directly to the ANS motif. These data suggest that directed nuclear localization and compartmentalization of the core complex of the apoB RNA editing enzyme is regulated through a dominant targeting sequence (ANS) contained within ACF.

Highlights

Apolipoprotein B1 mRNA is the target of a site-specific deamination reaction that introduces a C to U change in the edited transcript [1]
A mutant apobec-1 complementation factor (ACF) construct with an internal deletion spanning residues 360 – 401 showed a cytoplasmic, perinuclear localization pattern when expressed in COS-7 cells
We demonstrate that ACF shuttles between the nucleus and cytoplasm and that this shuttling likely involves at least two candidate transport proteins, transportin 2 and CRM1

Summary

Introduction

Apolipoprotein B (apoB) mRNA is the target of a site-specific deamination reaction that introduces a C to U change in the edited transcript [1]. These include apobec-1, an RNAspecific cytidine deaminase that represents the catalytic subunit of the core enzyme complex and which binds to a competence factor, ACF, that likely functions as the RNA binding subunit [2, 3] Physical interaction of these two components is required in order for this minimal complex to mediate C to U RNA editing of a synthetic apoB transcript in vitro [4, 5]. Co-expression of an ACF mutant that lacks the apobec-1 interaction domain along with wild-type apobec-1 reveals that apobec-1 remains cytoplasmic, whereas the ACF mutant localizes to the nucleus [4] These observations raise the possibility that, in addition to its requisite function in the enzymatic catalysis of C to U deamination, ACF may regulate apoB mRNA editing by controlling nuclear accumulation of apobec-1 through proteinprotein interactions and directed intracellular trafficking. The function of this motif and the regulation of nuclear-cytoplasmic transport of ACF and the ACF-apobec-1 complex is the focus of this report

Methods

Results

Conclusion