Importin α/β Mediates Nuclear Transport of a Mammalian Circadian Clock Component, mCRY2, Together with mPER2, through a Bipartite Nuclear Localization Signal

Yoko Sakakida,Yoichi Miyamoto,Emi Nagoshi,Makoto Akashi,Takahiro J Nakamura,Takayoshi Mamine,Megumi Kasahara,Yasuhiro Minami,Yoshihiro Yoneda,Toru Takumi

doi:10.1074/jbc.m413236200

Abstract

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin alpha/beta system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin alphas, alpha1 (Rch1), alpha3 (Qip-1), and alpha7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin alpha/beta system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.

Highlights

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks
These results suggest that the importin ␣/␤ system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes
Nuclear localization signals (NLSs) in mouse PER1, rat PER2, zebra fish CRY1, and Xenopus CRY1 have so far been identified (20 –23); it is still unclear whether nuclear entry is achieved via importin/karyopherin binding to NLS within the clock molecules or via other mechanisms

Summary

The abbreviations used are

CKI⑀, casein kinase I⑀; NLS, nuclear localization signal; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; DTT, dithiothreitol; MBP, maltose-binding protein; IBB, importin ␤ binding domain of importin ␣; WT, wild type; CKI, casein kinase I; Ran, Ras-related nuclear protein; MT, mutant. Nuclear localization signals (NLSs) in mouse PER1 (mPER1), rat PER2 (rPER2), zebra fish CRY1 (zCRY1), and Xenopus CRY1 (xCRY1) have so far been identified (20 –23); it is still unclear whether nuclear entry is achieved via importin/karyopherin binding to NLS within the clock molecules or via other mechanisms. A small GTPase Ran ensures the direction of nuclear transport by regulating the interaction between the receptors and their cargoes through its GTP/GDP cycle. Alternative nuclear import pathways have been described in which the NLS-containing proteins directly bind one of the nuclear import receptors of the importin-␤ superfamily without interaction with importin ␣ [27,28,29]. Examples of Ran and energy- or importin-␤-independent nuclear transport mechanisms have been reported [30, 31]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION