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S1 Fig. NLS function of the Xenopus KR sequence and Mm_Bt. (A) Subcellular localization
of GST-mRFP fusion constructs for the Xenopus KR sequence. Upper panel, schematic representation
of GST-mRFP fusion constructs. KRa, KRb, and KRm were derived from the KR of
Xl_Nemp1a, Xl_Nemp1b, and the corresponding region of Mm_Nemp1, respectively. KRa
(δR) is a deletion mutant of KRa. Lower panels, subcellular localization of GST-mRFP fusion
constructs. COS-7 cells were transfected with HA-tagged GST-mRFP fusion constructs as indicated,
fixed, and stained with anti-HA antibody (red) and SYTOX Green for DNA. Scale bars,
5 μm. (B) Subcellular localization of Mm_Bt and its GST-mRFP- HA construct. COS-7 cells
were transfected with the HA-tagged mouse Bt construct (Mm_Bt-HA) or GSTmRFP-
Mm_Bt-HA, fixed, and stained with anti-HA antibody (red) and SYTOX Green for
DNA. GST-mRFP-Mm_Bt-HA exhibited cytoplasmic localization, but also nuclear localization
in some cases. Scale bars, 5 μm. We have previously shown that Myc-tagged Xl_Ct and KR constructs
but not Xl_Bt (see Fig 1A) is localized to the nucleus, suggesting NLS function of the
KR sequence [10]. Therefore, we systematically examined the nuclear localization activity of
KR, using GST-mRFP-HA, which alone cannot be transported into the nucleus because of its
large molecular mass (122 kDa as a dimer under the native conditions). GST-mRFP-HA was
fused with short peptides related to the KR sequence, and the fusion constructs were analyzed
for their ability to localize to the nucleus. As shown in S1 Fig A, GST-mRFP-HA alone was localized
in the cytoplasm, whereas the SV40NLS fusion, which served as a positive control, exhibited
nuclear localization. Similarly, the KR fusion proteins, KRa and KRb, which were
derived from the Xenopus homoeologs of Nemp1, Nemp1a and Nemp1b, exhibited nuclear localization,
whereas the KRa(δR) fusion did not, indicating that both KRa and KRb sequences
function as NLSs (S1 Fig B) and that the first Arg residue of the RKIKXKRAK (X is R or L)
motif is required for this activity. We also analyzed a short sequence from Mm_Nemp1, whose
position corresponds to that of KR in Xl_Nemp1, named KRm, though KRm does not contain
a canonical NLS sequence. As expected, KRm did not elicit NLS function (S1 Fig A). However,
although Mm_Bt does not have a canonical NLS sequence either, HA-tagged Mm_Bt exhibited
nuclear localization (S1 Fig B; upper panels). Therefore, we analyzed NLS function of Mm_Bt
suing GST-mRFP-HA, and observed that GST-mRFP-Mm_Bt-HA exhibited weak nuclear localization
(middle panels, compare with GST-mRFP-HA negative control), and, in a few cases,
it was exclusively localized to the nucleus (lower panels), suggesting that Mm_Bt could have
NLS function. Thus, we conclude that the C terminal region of Nemp1 proteins (that is, KR in
Xenopus and Bt in mouse) exhibits NLS function.
(TIF) Image published in: Shibano T et al. (2015) Image reproduced on Xenbase with permission of the publisher and the copyright holder. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |