Identifying the nuclear envelope receptor of Mto1 in fission yeast Schizosaccharomyces pombe

Abstract

Microtubules are essential components of eukaryotic cytoskeleton and play a crucial role in variety of cell activities, such as cell mobility, intracellular transportation, cell division and organelle spatial organization. The initiation of microtubule nucleation is an important event to trigger the microtubule growth from different microtubule organizing centres (MTOCs) during the cell cycle in fission yeast. In interphase, the major MTOCs in the cells are nuclear envelope (NE) and microtubules existing in the cytoplasm. During mitosis, spindle pole body (SPB) that is the centrosome equivalent is the MTOC where astral and spindle microtubules initiate from. Once cells enter anaphase, the post-anaphase array (PAA) of microtubules will initiate from the equatorial MTOC (eMTOC) at the cell division site. To initiate microtubule nucleation, γ-tubulin small complex (γ-TuSC) will be recruited to the MTOC to form the “lock-washer” like γ-tubulin ring complex (γ- TuRC) as the scaffold of the microtubule. Fission yeast γ-TuSC is composed of two molecules of γ-tubulin and one each of GCP2 and GCP3 homologue, Alp4 and Alp6, respectively. Apart from Alp4 and Alp6, the homologue of human GCP4, GCP5 and GCP6, named Ghf1, Mod21 and Alp16 were identified as independent components of γ-TuRC in fission yeast. Protein Mto1 form a complex with its partner Mto2 and the complex directly interacts with γ-TuSC at all the MTOCs through the cell cycle. Mto1 is a large coiled-coil protein composed of 1115 amino acids. It has three main functional domains, including an N-terminal ~60 amino acids region termed CM1 motif that is required for the recruitment γ-TuSC to MTOCs, a central region that is required for the interaction with Mto2 at all cytoplasmic MTOCs and a ~44 amino acids region close to the C-terminus (named MASC) which is required for the binding of Mto1 to SPBs and eMTOC. The C-terminal truncation for Mto1 shows Mto1[1-549]-GFP mainly localizes on the NE and this Mto1 mutant is still functional for microtubule nucleation. It is referred as “Mto1[NE]”. In addition, truncation of 1-130 amino acids region for Mto1[1-549]-GFP creates the smallest Mto1 mutant that is able to initiate the microtubule nucleation in cytoplasm in a random manner. This Mto1[131-549]-GFP mutant fails to localize at any MTOCs, including NE. It is then referred as “Mto1[bonsai]”. To understand the mechanism that how is Mto1[NE] recruited to the NE, I performed two-step purification and mass spectrometry for both Mto1[1-9A1-549] and Mto1[131-9A1-549] strains, which form more significant puncta on both NE and cytoplasm respectively, to identify the potential receptor(s) of Mto1[1-9A1-549] on the nuclear envelope by comparing the different interactomes of Mto1[1-9A1-549] and Mto1[131-9A1-549]. Here, I show both exportin Crm1 and nucleoporin Nup146 are essential for the binding of Mto1 to the NE. I find the Localization of Mto1[1-9A1-549] GFP on the NE depends on binding of Mto1 to Crm1 via a nuclear export signal (NES)-like sequence within the N-terminus of Mto1. Further, I figure out that Spi1GTP (RanGTP in fission yeast) is involved in the formation of Crm1-Spi1GTP-Mto1 complex and required for the interaction of Mto1 to the NE. In addition, I also find that the FG repeats of Nup146 are essential for the binding of Mto1 to the nuclear envelope. It’s likely Nup146 anchors Crm1-Spi1GTP-Mto1 complex to the NE through the interaction between its FG repeats and Crm1. Last, my data are consistent with previous findings that the association of Mto1 to the NE is important for the microtubule nucleation from the NE.