The N-terminal TOG domain of Arabidopsis MOR1 modulates affinity for microtubule polymers

Abstract

SummaryMicrotubule-associated proteins of the highly conserved XMAP215/Dis1 family promote both microtubule growth and shrinkage, and move with the dynamic microtubule ends. The plant homologue, MOR1, is predicted to form a long linear molecule with five N-terminal TOG domains. Within the first (TOG1) domain, the mor1-1 leucine to phenylalanine (L174F) substitution causes temperature-dependent disorganization of microtubule arrays and reduces microtubule growth and shrinkage rates. By expressing the two N-terminal TOG domains (TOG12) of MOR1, both in planta for analysis in living cells and in bacteria for in vitro microtubule-binding and polymerization assays, we determined that the N-terminal domain of MOR1 is crucial for microtubule polymer binding. Tagging TOG12 at the N-terminus interfered with its ability to bind microtubules when stably expressed in Arabidopsis or when transiently overexpressed in leek epidermal cells, and impeded polymerase activity in vitro. In contrast, TOG12 tagged at the C-terminus interacted with microtubules in vivo, rescued the temperature-sensitive mor1-1 phenotype, and promoted microtubule polymerization in vitro. TOG12 constructs containing the L174F mor1-1 point mutation caused microtubule disruption when transiently overexpressed in leek epidermis and increased the affinity of TOG12 for microtubules in vitro. This suggests that the mor1-1 mutant protein makes microtubules less dynamic by binding the microtubule lattice too strongly to support rapid plus-end tracking. We conclude from our results that a balanced microtubule affinity in the N-terminal TOG domain is crucial for the polymerase activity of MOR1.

Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco

“…S4), indicating that TOG1 and TOG2 of MOR1 might bind unpolymerized tubulin. However, the N-terminal part of MOR1 containing TOG1 and TOG2 bound sheep brain tubulin very weakly when analyzed by gel filtration (Lechner et al, 2012). We also found that our MOR1 TOG column, containing TOG1 and TOG2, does not bind plant tubulin efficiently.…”

Drosophila melanogaster Mini Spindles TOG3 Utilizes Unique Structural Elements to Promote Domain Stability and Maintain a TOG1- and TOG2-like Tubulin-binding Surface

“…In the case of the XMAP215 family members, mutating tubulin binding activity in any subset of TOG domains within the array affects tubulin binding and MT polymerization activity with concomitant effects on mitotic spindle size (8,16,17,20,29,42,43). The key to understanding the XMAP215 TOG array MT polymerization mechanism is determining how many tubulin heterodimers the array binds, whether the pentameric TOG array simply tethers tubulin heterodimers or actively potentiates longitudinal or lateral tubulin-tubulin interactions, and what nucleotide and/or conformational state of tubulin each TOG domain in the array preferentially binds.…”

“…Mutating the tubulin binding activity of different TOG domains in the pentameric TOG domain array has differential effects on XMAP215 MT polymerization activity and spindle length. Mutations in the TOG1 or TOG2 intra-HEAT tubulin-binding loops compromise XMAP215-mediated MT polymerization activity more dramatically than homologous mutations in TOG3 or TOG4 (8,16,17,20,29,42,43). Mutating the tubulin-binding determinants in TOG5 does not appear to significantly affect the rate of MT polymerization (16,29).…”

The Microtubule Plus-End Tracking Protein ARMADILLO-REPEAT KINESIN1 Promotes Microtubule Catastrophe in Arabidopsis

“…This observation possibly resulted from increased resistance to microtubule destabilizing drugs and bundling typically associated with the 35S pro :GFP-MBD marker (Marc et al, 1998;Lechner et al, 2012). (A) Plus-end velocities of EB1b-GFP of CMTs and EMTs were measured in wild-type and ark1-1 elongating root hairs upon imaging with spinning-disc confocal microscopy.…”