Abstract
Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.
Highlights
Non-centrosomal microtubule organizing centers (MTOCs) are critical to the morphology and function of many types of cells (Petry and Vale, 2015; Sanchez and Feldman, 2017; Wu and Akhmanova, 2017), especially cells in which interphase microtubules (MTs) are arranged in linear rather than radial arrays (Bartolini and Gundersen, 2006)
Using a comparative-interactome mass spectrometry approach, we find that nuclear envelope (NE) localization depends on the Mto1 N-terminus interacting with exportin Crm1, a nuclear transport receptor, and nucleoporin Nup146, a component of the nuclear pore complex (NPC)
Nuclear positioning was less accurate in mto1[bonsai]-GFP and mto1[D130]-GFP cells compared to mto1[NE]-GFP and mto1-GFP cells, indicating that MT nucleation from the NE contributes to nuclear positioning
Summary
Non-centrosomal microtubule organizing centers (MTOCs) are critical to the morphology and function of many types of cells (Petry and Vale, 2015; Sanchez and Feldman, 2017; Wu and Akhmanova, 2017), especially cells in which interphase microtubules (MTs) are arranged in linear rather than radial arrays (Bartolini and Gundersen, 2006). Bao et al have devised a new method to identify the proteins that recruit Mto to the surface of the nucleus. Sites of non-centrosomal g-tubulin complex recruitment include pre-existing microtubules themselves, as well as membrane-bound compartments such as the Golgi apparatus and the nuclear envelope (NE). Combined recruitment of g-tubulin complex and MT minus-end stabilizers/anchoring proteins is important for MTOC organization at the cell cortex in diverse types of epithelial cells (summarized in [Sanchez and Feldman, 2017; Wu and Akhmanova, 2017]). In addition to revealing the mechanism of MTOC formation at the fission yeast NE, our work demonstrates a completely novel role for the nuclear export machinery, in which the exportin is repurposed to create NPC-docking sites for cytoplasmic proteins with functions unrelated to nuclear export
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