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Abstract Spindle assembly in vertebrates requires the Aurora kinase, which is targeted to microtubules and activated by TPX2 (Targeting Protein of XKLP2). In Arabidopsis (Arabidopsis thaliana), TPX2-LIKE 3 (TPXL3), but not the highly conserved TPX2, is essential. To test the hypothesis that TPXL3 regulates the function of α Aurora kinase in spindle assembly, we generated transgenic Arabidopsis lines expressing an artificial microRNA targeting TPXL3 mRNA (amiR-TPXL3). The resulting mutants exhibited growth retardation, which was linked to compromised TPXL3 expression. In the mutant cells, α Aurora was delocalized from spindle microtubules to the cytoplasm, and spindles were assembled without recognizable poles. A functional TPXL3-GFP fusion protein first prominently appeared on the prophase nuclear envelope. Then, TPXL3-GFP localized to spindle microtubules (primarily toward the spindle poles, like γ-tubulin), and finally to the re-forming nuclear envelope during telophase and cytokinesis. However, TPXL3 was absent from phragmoplast microtubules. In addition, we found that the TPXL3 N-terminal Aurora-binding motif, microtubule-binding domain, and importin-binding motif, but not the C-terminal segment, were required for its mitotic function. Expression of truncated TPXL3 variants enhanced the defects in spindle assembly and seedling growth of amiR-TPXL3 plants. Taken together, our findings uncovered the essential function of TPXL3, but not TPX2, in targeting and activating α Aurora kinase for spindle apparatus assembly in Arabidopsis.
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Protein salvage and repurposing in evolution: Phospholipase D toxins are stabilized by a remodeled scrap of a membrane association domain
Abstract The glycerophosphodiester phosphodiesterase (GDPD)‐like SMaseD/PLD domain family, which includes phospholipase D (PLD) toxins in recluse spiders and actinobacteria, evolved anciently in bacteria from the GDPD. The PLD enzymes retained the core (β/α)8barrel fold of GDPD, while gaining a signature C‐terminal expansion motif and losing a small insertion domain. Using sequence alignments and phylogenetic analysis, we infer that the C‐terminal motif derives from a segment of an ancient bacterial PLAT domain. Formally, part of a protein containing a PLAT domain repeat underwent fusion to the C terminus of a GDPD barrel, leading to attachment of a segment of a PLAT domain, followed by a second complete PLAT domain. The complete domain was retained only in some basal homologs, but the PLAT segment was conserved and repurposed as the expansion motif. The PLAT segment corresponds to strands β7–β8 of a β‐sandwich, while the expansion motif as represented in spider PLD toxins has been remodeled as an α‐helix, a β‐strand, and an ordered loop. The GDPD‐PLAT fusion led to two acquisitions in founding the GDPD‐like SMaseD/PLD family: (1) a PLAT domain that presumably supported early lipase activity by mediating membrane association, and (2) an expansion motif that putatively stabilized the catalytic domain, possibly compensating for, or permitting, loss of the insertion domain. Of wider significance, messy domain shuffling events can leave behind scraps of domains that can be salvaged, remodeled, and repurposed.
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- PAR ID:
- 10426638
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 32
- Issue:
- 7
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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