Identification and mutation of a nucleolar
localization signal (NoLS) in coilin. (A) Coilin contains a sequence,
located between the two putative NLSs, that is nearly identical to the
NoLS of MDM2 (Lohrum et al., 2000). Stippled boxes
indicate potentially-acidic serine patches (AC) spanning amino acids
242–259 and 312–325. Facing page: (B) The putative coilin NoLS was
mutated from KKNKRKNK to IINNIINI (mt NoLS) in both the GFP-coilin and
GFP-coilin(1–248) backgrounds. Note that, while the truncation alone
localized to nucleoli and CBs, mutation of the NoLS depleted coilin
from nucleoli. (C) Similarly, block substitution of the two acidic
patches (S245–252A and S314–320A, termed GFP-coilin(mt AC)) also
resulted in nucleolar accumulation. Additional localization in CBs was
occasionally observed (our unpublished results). However, mutant
proteins bearing substitutions in both the NoLS and the acidic patches
(GFP-coilin(mt AC; mt NoLS)) did not accumulate in nucleoli, resulting
in a distribution pattern indistinguishable from that of wild-type
coilin.