The <em>Aspergillus nidulans</em> Kinesin-3 Tail Is Necessary and Sufficient to Recognize Modified Microtubules

<div><p>Posttranslational microtubule modifications (PTMs) are numerous; however, the biochemical and cell biological roles of those modifications remain mostly an enigma. The <em>Aspergillus nidulans</em> 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 <em>A. nidulans</em> 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.</p> </div

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

<p>(<b>A</b>) 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>B</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). (<b>C</b>) 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.</p

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.

<p>Hyphae are 3 µm in diameter.</p

<i>A. nidulans</i> strains used in this study.

<p><i>A. nidulans</i> strains used in this study.</p

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

<p>(<b>A</b>) 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 (<i>K</i>) 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>B–E</b>) Localization of different UncA truncated versions (as indicated) in the <i>ΔuncA</i> strain SNZ9. UncA proteins were labeled with GFP and expressed under the control of the <i>uncA</i> promoter. Scale bar, 5 µm. (<b>F</b>) Confirmation of expression levels by Western blot analysis of GFP-UncA^rigor (206 kDa)(SNZ14) and GFP-UncA^rigor<i>Δ</i>1316–1402 (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. (<b>G</b>) Colonies of SNZ9, SCoS75 and wildtype (TN02A3). (<b>H</b>) Alignment of the 86 aa region of UncA orthologues from different fungi and higher eukaryotes. Done with CLC Sequence Viewer 6. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030976#pone.0030976.s001" target="_blank">Figure S1</a>. (<b>I</b>) Calculation of isoelectric points for distinct regions of UncA.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

LC/MS analysis of metabolites produced by <i>Alternaria alternata</i> WT, WT transformed with an empty vector (E), the Δ<i>pksJ</i> strains, the RNAi-<i>pksJ</i> strain, the <i>ΔpksH</i> and the RNAi-<i>pksH</i> strains as detected by UV absorbance.

<p>m/z values are given for the most prominent peaks. Peak no 5 corresponds to alternariol and peak no 11 correspond to alternariol-9-methyl ether.</p

Architecture of PKSs of <i>Alternaria alternata</i>.

<p>KS, β-ketoacyl synthase; AT, acyltransferase; DH, dehydratase; MT, methyltransferase; ER, enoyl reductase; KR, ketreductase; ACP, acyl carrier protein, CD, condensation domain; AA, Amino acid adenylation domain; CS:Chalon- and Stilben-Synthase (N)/(C); UDG: Uracil DNA Glycolase Superfamily; NAD, NAD binding domain. The sequences of the PKS loci are deposited under the following accession numbers: <i>pksA</i> (JX103636); <i>pksB</i> (JX103637); <i>pksC</i> (JX103638); <i>pksD</i> (JX103639); <i>pksE</i> (JX103640); <i>pksF</i> (JX103641); <i>pksG</i> (JX103642); <i>pksH</i> (JX103643); <i>pksI</i> (JX103644); <i>pksJ</i> (JX103645). The genbank accession numbers are given in brackets.</p

LC/MS analysis of alternariol (AOH) and alternariol-9-methyl ether (AME) formation in wildtype (WT), empty vector (E) and RNAi transformant of selected genes flanked by <i>pksJ</i> and <i>pksH</i>.

<p>H-TF:PksH-transferase; H-CL: PksH-cyclin F-Box; H-cP450: PksH-cytochrome p450; <i>altR</i> ( = alternariol regulator). AOH and AME values are expressed in nmol/10 µl.</p

Time-course expression analysis of different PKS genes.

<p>(<b>A</b>) TLC analysis of AOH formation at different time points. Extracts from cultures grown on MCDB agar at 28°C in constant darkness for 3, 5, 7, 10, 12 and 14 days, respectively. For each time point three extractions were performed. The last lane contained the AOH standard. (<b>B</b>) Quantitative real time reverse-transcription polymerase chain reaction (RT-PCR) gene expression analysis of different PKS genes after 7, 12 and 14 days of post inoculation (dpi).</p