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  • Separable roles for RanGTP in nuclear and ciliary trafficking of a kinesin-2 subunit.

Separable roles for RanGTP in nuclear and ciliary trafficking of a kinesin-2 subunit.

The Journal of biological chemistry (2020-11-26)
Shengping Huang, Larissa L Dougherty, Prachee Avasthi
ABSTRACT

Kinesin is part of the microtubule-binding motor protein superfamily, which serves important roles in cell division and intraorganellar transport. The heterotrimeric kinesin-2, consisting of the heterodimeric motor subunits, kinesin family member 3A/3B (KIF3A/3B), and kinesin-associated protein 3 (KAP3), is highly conserved across species from the unicellular eukaryote Chlamydomonas to humans. It plays diverse roles in cargo transport including anterograde (base to tip) trafficking in cilia. However, the molecular determinants mediating trafficking of heterotrimeric kinesin-2 itself are poorly understood. It has been previously suggested that ciliary transport is analogous to nuclear transport mechanisms. Using Chlamydomonas and human telomerase reverse transcriptase-retinal pigment epithelial cell line, we show that RanGTP, a small GTPase that dictates nuclear transport, regulates ciliary trafficking of KAP3, a key component for functional kinesin-2. We found that the armadillo-repeat region 6 to 9 (ARM6-9) of KAP3, required for its nuclear translocation, is also necessary and sufficient for its targeting to the ciliary base. Given that KAP3 is essential for cilium formation and there are the emerging roles for RanGTP/importin β in ciliary protein targeting, we further investigated the effect of RanGTP in cilium formation and maintenance. We found that precise control of RanGTP levels, revealed by different Ran mutants, is crucial for cilium formation and maintenance. Most importantly, we were able to provide orthogonal support in an algal model system that segregates RanGTP regulation of ciliary protein trafficking from its nuclear roles. Our work provides important support for the model that nuclear import mechanisms have been co-opted for independent roles in ciliary import.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cycloheximide, Biotechnology Performance Certified
Sigma-Aldrich
Importazole, ≥98% (HPLC)
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Monoclonal Anti-Tubulin, Acetylated antibody produced in mouse, clone 6-11B-1, ascites fluid