The Aspergillus nidulans kinesin-3 tail is necessary and sufficient to recognize modified microtubules

Abstract

Posttranslational microtubule modifications (PTMs) are numerous; however, the biochemical and cell biological roles of those modifications remain mostly an enigma. The Aspergillus nidulans kinesin-3 UncA uses preferably modified microtubules (MTs) as tracks for vesicle transportation. Here, we show that a positively charged region in the tail of UncA (amino acids 1316 to 1402) is necessary for the recognition of modified MTs. Chimeric proteins composed of the kinesin-1 motor domain and the UncA tail displayed the same specificity as UncA, suggesting that the UncA tail is sufficient to establish specificity. Interaction between the UncA tail and alpha-tubulin was shown using a yeast two-hybrid assay and in A. nidulans by bimolecular fluorescence complementation. This is the first demonstration of how a kinesin-3 motor protein distinguishes among different MT populations in fungal cells, and how specificity determination depends on the tail rather than the motor domain, as has been demonstrated for kinesin 1 in neuronal cells.

Figure 1. UncA-deletion analysis reveals that the tail of UncA is involved in specificity determination.

(A) Scheme for the UncA-deletion analysis. The number of amino acids is given in front of the truncated proteins. Motor = motor domain containing the lysine-rich loop (K) and a rigor mutation in the P-loop (asterisk); FHA = forkhead associated domain; PH = pleckstrin homology domain. The red square indicates a 86 aa amino acid stretch. (B–E) Localization of different UncA truncated versions (as indicated) in the ΔuncA strain SNZ9. UncA proteins were labeled with GFP and expressed under the control of the uncA promoter. Scale bar, 5 µm. (F) Confirmation of expression levels by Western blot analysis of GFP-UncA^rigor (206 kDa)(SNZ14) and GFP-UncA^rigor Δ1316–1402 (194 kDa)(SCoS124). Western blot detection was done with anti-GFP antibodies (1∶4000) and anti-rabbit IgG peroxidase conjugated secondary antibodies (1∶4000). 285 ng crude protein extract was loaded. (G) Colonies of SNZ9, SCoS75 and wildtype (TN02A3). (H) Alignment of the 86 aa region of UncA orthologues from different fungi and higher eukaryotes. Done with CLC Sequence Viewer 6. See also Figure S1. (I) Calculation of isoelectric points for distinct regions of UncA.

Figure 2. Analysis of UncA versions with deletions of the forkhead associated domain (FHA)(SCoS61), the pleckstrin homology domain (PH)(ScoS16), and the coiled coils (CC)(SCoS81) region.

Hyphae are 3 µm in diameter.

Figure 3. Chimeric kinesin proteins verify that the UncA tail is sufficient for microtubule specificity.

(A) Scheme for the creation of chimera of kinesin 1 (yellow), KinA, and kinesin 3, UncA (green). (B) Localization of GFP-UncA (SNZ2) and (C) mRFP-KinA (SCS6-NZ). (D) Time lapse of the KinA–UncA chimeric protein (SCoS23). Arrows indicate a moving vesicle. Vesicles also accumulate at the tip of hyphae, similar to the UncA localization. (E) Growth comparison of WT, ΔuncA and the ΔuncA strain complemented with the KinA-UncA chimera (SCoS23). The fusion protein can restore the ΔuncA phenotype. (F) Localization pattern of KinA^rigor-UncA chimera (SCoS24) labeled with GFP in the ΔuncA strain, under the control of the uncA promoter. The chimera shows the same specificity as UncA^rigor. (G) In contrast UncA^rigor-KinA chimera (SCoS44) in ΔuncA, labeled with GFP, under the control of the uncA promoter do not label MT subpopulations. Hyphae are 3 µm in diameter.

Figure 4. The tail of UncA is able to bind to alpha tubulin.

(A) Yeast two-hybrid interaction tests with different truncations of UncA to map the interaction site between these proteins. Only the full-length tail region of UncA is able to interact with either of the two alpha tubulins. Transformants were assayed for growth on SD-LW to confirm integration of both constructs (left) and on SD-QDO for nutritional selection for positive interactions (right). The strength of the interaction is shown in the X-α-Gal assay. The red square indicates the 86 amino acids region. (B) Bimolecular fluorescence complementation assay with the YFP-C-terminal half fused to the UncA-tail and the YFP-N-terminal half fused to TubA in strain (SCoS126). (C) Subcellular localization of the GFP-UncA-tail in SCoS127. The tail of UncA localizes to vesicles, which moved in antero- and retrograde direction. Hyphae are 3 µm in diameter.