APOBEC3B and AID have similar nuclear import mechanisms - PubMed (original) (raw)

APOBEC3B and AID have similar nuclear import mechanisms

Lela Lackey et al. J Mol Biol. 2012.

Abstract

Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.

Copyright © 2012 Elsevier Ltd. All rights reserved.

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Figures

Figure 1

Figure 1. Relationships between AID and A3B

(a) Phylogenetic tree depicting the APOBEC loci in the indicated species. The split between fish and birds (~300 million years ago) and the divergence of the original placental mammal (~100 million years ago) are shown,,. (b) Representative images of HeLa cells transfected with human or zebrafish AID-eGFP and treated with lepB or ethanol as a vehicle control. (c) Representative images of HEK293T or HeLa cells expressing human AID-eGFP after treatment for the indicated times with lepB. (d) Representative images of HEK293T or HeLa cells expressing A3B-eGFP (quantified below; mean and SD shown for >20 individual cell measurements). (e) Representative images of A3B-eGFP expressed in the buccal tumor epithelial line TR146, the squamous cell carcinoma line JSQ3, the breast epithelial line MCF10A, and the osteosarcoma line U2OS.

Figure 2

Figure 2. A3B is actively imported into the nucleus

(a) Representative images of digitonin treated HeLa cells incubated with lysates containing GFP, A3B-eGFP, A3B NTD-eGFP, A3A-eGFP, or A3A NLS-eGFP. White arrows highlight instances of active nuclear import. (b) Quantification of the results from (a) using the same exposure for all conditions (FITC=1 second; n≥30 cells were examined and the mean nuclear fluorescent intensity is indicated for each condition). The inset is an anti-GFP immunoblot of representative cell lysates with asterisks indicating the correct bands.

Figure 3

Figure 3. A3B and AID localize differently during the mitosis

Representative image frames of (a) mCherry, (b) A3B-mCherry, and (c) AID-mCherry localization during HeLa cell cycle progression from late telophase of mitosis to early interphase. H2B-eGFP images of the same cells are shown below each time series. The white arrows at 0 min highlight informative cells, and the arrows at other time-points indicate significant localization events discussed in the text.

Figure 4

Figure 4. Single amino acid changes within the β2 region affects nuclear localization of A3B and AID

(a) Representative images of A3B-eGFP and A3B V54D-eGFP in HeLa cells. The adjacent dot plot reports quantification of the nuclear to total fluorescent signal (n≥25 cells were analyzed for each condition with mean and SD shown). (b) Representative images of AID-eGFP and AID Y48H-eGFP in HeLa cells taken 3 hrs after ethanol or lepB treatment. The adjacent dot plot reports quantification of the nuclear to total fluorescent signal (n≥50 cells were analyzed for each condition with mean and SD shown). Model ribbon structures of AID and A3B NTD depicted adjacent to the actual structure of A3G C-terminal domain (CTD). The β2 region and key amino acids in AID and A3B are labeled in red and circled; the side chains of zinc-coordinating residues are illustrated in green.

Figure 5

Figure 5. Both AID and A3B interact with members of the adaptor importin family

Immunoblots of input HEK293T protein lysates (bottom panel) and pulldown results for eGFP, AID-eGFP, AID Y48H-eGFP, A3B-eGFP and A3B V54D-eGFP with the indicated GST-importin substrates (top panels).

Figure 6

Figure 6. A3B does not perform class switch recombination and AID does not restrict HIV-1

(a) Flow cytometry quantification of the isotype switch to IgG in AID-deficient murine B cells expressing AID, A3B or non-functional mutants (mean and SD are shown for duplicate samples). (b) Infectivity of HIV-1 produced in HeLa cells expressing HA tagged APOBEC3G (A3G), A3B, AID and their catalytic mutants. High levels of restriction correspond to lower levels of fluorescence in a reporter CEM-GFP cell line and a decrease in infectious virus. Asterisks indicate that twice as much DNA was required for adequate expression of AID (0, 50, 100 and 200 ng). (c) Immunoblots of viral particle proteins (top) or cell lysate proteins (bottom) probed for anti-HA (APOBEC/AID expression), anti-p24 for a virus loading control, or anti-tubulin for a cellular loading control. (d) Representative images of mCherry, A3B-mCherry or AID-mCherry in HeLa cells 24 hrs after infection with replication competent HIV-1LAI nef::eGFP. Cells expressing the protein of interest infected with HIV are indicated by white arrows.

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