Download A non-conventional nuclear import pathway Sandra Korge1, Bert

A non-conventional nuclear import pathway Sandra Korge1, Bert Maier1,
Achim Kramer1 1Charité-Universitätsmedizin Berlin, Berlin, GERMANY
Generating a 24 hour rhythm of the molecular circadian clock is influenced
by transcriptional and translational regulation as well as post-translational
processes as nucleocytoplasmic protein shuttling. As it is known for Period
(PER), Cryptochrome (CRY) and other clock proteins to carry classical
nuclear localization signals (NLS) little is known about Tnpo1 interacting
domains. Using RNAi, we could show period lengthening upon knockdown
of importin beta (Imp ? ), which recognizes classical NLS. On the opposite
knockdown of transportin1 (Tnpo1), the M9 interacting carrier, results in
period shortening. Therefore we investigated potential target clock
proteins bioinformatically for M9 characteristic amino acid sequences such
as the PY motif. We found potential M9-like sequences in BMAL1, PERs and
CRYs as well as in Casein kinases and RevErb?. To study the influence of
TNPO1 on clock protein localization, we performed a nuclear import assay,
where putative M9-sequences of clock proteins are fused to a cyan
fluorescent protein (CFP). The M9-CFP fusion protein will change its
localization from cytoplasmic to nuclear, if the putative M9 sequence is
sufficient to interact with a nuclear import carrier, potentially TNPO1. To
clarify if the M9-CFP fusion protein is actively translocated by TNPO1,
nuclear import was monitored upon Tnpo1 knockdown. We identified
putative M9 sequences in CRY1, CRY2 as well as PER2 and were able to
show their specific TNPO1 translocation. Furthermore fluorescence
recovery after photobleach (FRAP) will give the opportunity to study the
kinetics of the M9-CFP nuclear import in the presence or absence of
TNPO1.