Tubulin is actively exported from the nucleus through the Exportin1/CRM1 pathway

K Schwarzerová,V Dostál,J Martinek,P Nick,E Bellinvia,A C Schmit,L Fischer,P Bokvaj,L Sikorová,L Libusová

doi:10.1038/s41598-019-42056-6

K Schwarzerová, V Dostál + Show 8 more

Open Access

https://doi.org/10.1038/s41598-019-42056-6

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Abstract

Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and β-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

Highlights

Introduction of point mutationmutNESβ2 or mutNESβ3 in full-length GFP-β-tubulin molecule fused to GFP resulted in dramatic loss of protein incorporation into microtubules (Supplementary information 2)
Based on our observations of nuclear tubulin accumulation in cells treated with leptomycin B, we suggest that the Exportin 1/CRM1 export pathway accounts for the majority of tubulin export from the nucleus, and we discuss the biological significance of tubulin compartmentalization into the cytoplasm during interphase
Four putative nuclear export sequences (NESs) were found in α-tubulins, and three in β-tubulin sequences of Arabidopsis thaliana

Summary

Introduction

MutNESβ2 or mutNESβ3 in full-length GFP-β-tubulin molecule fused to GFP resulted in dramatic loss of protein incorporation into microtubules (Supplementary information 2). Export activity conferred by tubulin NESs is inhibited by leptomycin B. We probed for the effect of Exportin 1/CRM1 inhibitor leptomycin B37,38 on the export activity of most active export signals expressed in BY-2 and U-2 OS cells. Leptomycin B treatment did not change nuclear:cytoplasmic signal ratio in cells expressing free GFP in U-2 OS (Fig. 3) and BY-2 (Fig. 4) cells

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