Analysis of the protein sequence

Results

Fig. IV.2: Detection of the kipB transcript and the KipB protein (A) Northern blot analysis of kipB. RNA was isolated from mycelia of FGSC26, incubated at 37°C, harvested after 16 h of vegetative growth, and hybridized to a kipB-specific probe. (B) Protein extracts (RMS011 (wt) and SPR3 (truncated KipB (690 aa.)-HA)) were subjected to Western blot analysis and probed with anti-HA antibodies. The asterisk at the right of the panel marks the position of the protein and the absence of the signal for the wt (FGSC26).

Results

The positions of these regions are conserved (DeZwaan et al., 1997), (West et al., 2001), and they can potentially mediate protein multimerization.

MGA- - S S DSS S I S VTVRVRP F TI REAAQLTKCEDGP L- -F- -LGDGS LAGAPAP KLN- - - -QKGLRSIIKVIDDRCLVF DP P EDNPV- - - -QKF MSGYP DP NAS S ITVAVRVRP F TI REAAQL- - - DEGTV- -F- -LGDGS LAAAPTP KLN- - - -QRGI RP VIKVVDDRCLVF DP P EDNPI - - - -QKF MS- - - - -RQS S ITVTVRVRP FS TAESANLI AS SDRLS - -F- - GTS SS LRNP GSGRQ- - - -I RRVVKVLDGRVLVF DP PDETTATLS AT- - - NRRL MK- - - EGS S I S VAVRVRP F TEREKGLLAETP KS KE- -F- -LGDGS LAVS NTS S NT- - - F CTNGI RKI VRVLDDNVLIF DP P EENP- - - -L MNV- P ETRQS S IVVAIRVRP F TS MEKTRLVNEASGAEANFP GLGDSS LI LP MSNNS DS DI DI DAEEGS TRS KRNS LLRRKVI RP EGI RKI VDCVDDRMLIF DPADRNPLNKVS DQVLNS MR S RS- -VVP N-GKRV- - - -KDQTF AF DRIF DQNATQGEVYEATTRSLLDS VLDGYNATVF AYGATGCGKTHTITGTP QQP GI I F LTMQELF ERI EERKSEKHTELS LSFLEI S RS- -VVP AMGKKV- - - -KDQVF AF DRIF DENAS QVEVYEGTTKGLLDS VLDGYNATVF AYGATGCGKTHTITGTP QSP GI I F LTMQELF EKINERS GEKHTEVTLS YLEI STS QQS LARLS RKS NNS AGF- - - GRDLRYAF DRVF DETATQQQVYERTARP LLDNILDGFNATIF AYGATGCGKTHTI S GTMQDP GLIYLTLKELF ERMDHLRDEKI F DLRLS YLEI AKVQKS LLP AGKR- - - -F- - - RDVRYAF DRLFGEEAS QEDVYKGTTEP LLDS VLQGYNATVF AYGATGCGKTHTI S GRPDDP GI I F LTMRALLDRVEGLKRTMNVDISVS YLEI ARATKATAS S I NNS NATNKFS S QRRRHGGEI KFVF DKLF DETSS QARVYKETTSP LLDS VLDGFNSTVF AYGATGCGKTYTVS GTPS QP GI I F LAMEELFNKITDLKDEKDFEIS LS YLEI YNETI RDLLVPGGA- - -KS -GLSLREDSNKAVS VSGLS S HS P KS VQEVMDMIMKGNACRTMS P TEANATS S RS HAVLQI NVAQKDRNADI NE- - - -P HTM-ATFS I I DLAGS ERAS ATK YNETI RDLLVP P GS ATNKQ-GLMLREDSNQGVS VAGLTS HKP KDVQEVMDMI VQGNEYRTVS P TAANAVS S RS HAVLQI NVAQKDRNAAVNE- - - -P HTM-ATLS I I DLAGS ERAS ATK YNETI RDLLVSPTPNQAKP- -LNLREDADRRI TVPGLTSLS PESLEEI IDI IMKGNANRTMS P TEANAAS S RS HAVLQVTLIQKPRTAGI NED- - - -HTL-ATLS I I DLAGS ERATATK YNEKI RDLLVQDP LSMEKPKSLNI CEDAEQNVS VPGLSYF TPTNLEEVMEI II RGNSNRTMS P TEANAVS S RS HAVLQIYI TQTP KS GEKQEES ES QNSHKVRS VFSFI DLAGS ERAS ATK YNERI RDLLKPETP S- - - - KRLVIREDTQNHI KVANLSYHHPNTVEDVMDLVVQGNINRTTS P TEANEVS S RS HAVLQIHI MQTNKLVDLTS Q- - - -HTF -ATLS I I DLAGS ERAAATR NRGERLF EGANI NKS LLALGSCI NALC- -DP RKRNHVP YRNS KLTRLLKF S LGGNCKTVMI VCVS P S SQHFDETQNTLRYANRAKNIQTKVTRNVFNVNRHVKDFLVKI DEQMALINELKA NRGERLLEGANI NKS LLALGSCI NALC- -DP RKSNHVP YRNS KLTRLLKF S LGGNCKTVMI VCVS P S S EHFDETQNTLRYANRAKNIQTKVTRNVFNVNRHVKDFLVKI DEQMALINELKA LRGSRLF EGANI NKS LLALGNCI NALC- -DPHRRAHVP YRDS KLTRLLKF S LGGNCRTVMI VCVS P S SVHYEETHNTLKYANRAKNI KTEVLRNMIS VDRHVSQYVKAIVELREQI S ELEN NRGKRLVEGANI NRS LLALGNCI NSLC- - EP RRRQHVP YRDS KLTRLLKF S LGGNCRTCMI VCIS P S S EHYDETHNTLKYGNRAKNI KTKVSRNVVS VDRHVSEYVRTIYELRQKVSILQK NRGIRLHEGANI NRS LLALGNCI NALCLNDGSRS CHIP YRDS KLTRLLKF S LGGNCKTVMI VCIS P S SSHYDETLNTLKYANRAKEI KTKI IRNQQSLSRHVGSYLKMITEQKRQIEELRE - - - - -QQRES EKVAFAKFKKQTEKKDAAVREGLARIRNAYDHS LPERQERI NNMI RLKQVS RRI GLLS S WI AAF DNVCAN- - - -S ENEVPLS NLQAVRKTAQGI LLELEGSRQHYHQRLAK - - - - -QQRDAEKVF FAKFLKQS EKRDAVVREGLARLHAAYEHAEKDRTEVIGLMKQQRAI ERRI GLLS S WI AAF DSVCDARGCAS EEEMP- AP LTTIRRTAVGI LNELES TRNHVHQRLGR RLAQI DLS S QS NGS DQDAVTQSF AHES KLAEARNLLRMTF EETLP LQNDTI NKVEKVKHF DDSIRVLKYWLS - - - - -CYERI LP NS ADERVF LVRS K- - - -LESLLTRRAEIIADI DP RIAE- - - -ESKQLALNKEVRKI S SREI KMLDARS MLKNS FDGSRDLQKS LIEHVRTLRRI EDEITLTKMWIS - - - IAKES DAMS GHNI KSVETR- - - -LAKLYDQRS LITAKVNP REEKM- - - I S LKLTKYKLNKEKI QLAI NECVNRVQQTYAGV- - - - -ETYQVAKTLKS LI - - - LCKRR- - - F LQMVKLEVDNLI LLF EREES TAAEMQP S TWDRGMTSAVENAVQQLQEF DTS D- - - KS DVTNLRREAELLRANTEREALS AVAEQDKAGD-AAVVQLLLQAHF EI ASS I ERI MHLSEEEAVEMGKRSLTKMLDS CCTATSNVVKP DS NL CNWERALDTALQHSI S QLP AMGGNSEGCNTERDILAREAELLRTGF MRDAYHEVVEQDKAGD-AAILQTLLMAQF EMMAS LEELLRMEERQAMEHAKAMVNRLLETGLQAVGQVVKP DGS L ELVYQKF QRSVS HI INTY- - - - -KQEGATMYADVLQDEVDLLKS I I ENQVLDA- - -QNKVDEFTPVLESLLRS SFKASS- - - -LLK- - - -EGGMQELFS ILEK- - - EEI CKTF QNS IS HIVSS F - - - - -KGEGADMYADMLQDDVDLLKS I I ENQILDA- - - KHES ETFS S TS RKLIQNLFL- - - -LFPLLP G- - - - - - VI SNCRMI SG- - - QLYNKI HELEMKF DETDTLSSVI HQVHS IDLN- - - -KLREMEDWDETYDLVY- -LES CLNQISELQRNE- - - P P MP TF S P S KHS P AKAKKR- - - LSLAI VP PS KSLNATVALHP TAP TSP - - - TRGSPRRRKMGTGR- KSVS F S P KKAPAKP- - - -P KRS VRWKDDE- EDGTLT P T- AGYQGTGLS VP KLRRKS GAI NGAKTIPQP MFS ASAALTTAI P TSTETAMDI S TEP EADVEGQLES WDGES VPHLSP VKARKVAYAS SKKAGTS F TFTP VKKKHS VRWRDDETEEGTLA - - - WLLGIGLGEKPNI S VLSESYKLNSTS DDSRTI NRD- - - RVHS FPTQP LLNNNLPRMF F V- - -KSPKKPVVFS KRS P KKRVRF DD- - - - - - NAI DVNES LARAF DQLVGIVP S EP TIQVP NLIEKG- - - K- - - AP LLS- - - - -MF EI - - - - -PRSP S RF KARS PSKAAR- - - - - - ILVNS S I MTE- - - KLMSD- - - -P GLNS RF KF LS- - - KWL- - - EI QKTP QKREATL- - I HRS VS PQEPGLPRAS P I P RGI P VP TRN- - - F S P S GGS S P I P TP S DQP LS I P KNNRFKTGF LS KK- - - -TGSSP- - - I P AP P TVALS VS DRS S - DF EKTP QKWDS S P GEP ENGI S PVRPP MPS YLGGMKS P P P P QRDNTDEDVEMDDGAGP ALS S I P DLS GLS I GKP NRFQAGF LS KS RTSLAGSTP GATI TGNGVLQS QP P P VF NLNLTNS NNS - - - -SMS TSDSGAS - - - - - - - MNRTPN- - - -IES I I QDLV- - - HI DEEF E- - - SFARTFI ANP DS NFTNTNI NI I NTTAADLAV- - - - - - -P LRDI EGS - - - S F LNRASTERP S RI AVRTP S GNY-S P SP AQP ETKGEWKAS KDD- - - VRRI STAM- - - -RRISI GS F GTSAS AT- - - I S QQNGGGEEKP TQP LRS I P VS RAANAYSS PIS YS AKTSNAAP STTNTMNS GNHDGS PKIP QPPTTS S LQQVDENQP P VTARKSSS RLS LGS AI PRRLSTSPRSGSGGS TDS DNSLVI DP L - - - -AYNS PI- - - QTS KLKNMNFF NTMHMP- - - S TPAHKRP ENKN- - - QIDVEI NLTS P VS P MLEDKPEPGL- - - -LI KSP L - - - V- - - LKKPLKKRVRFS - - EVP- - - TTSSVPP VEIKN- - - -K- - - DSKPK- - - -VEKSL - - - P AETLQR- - - QNFS- - - QKKVKWTS PDLS P S P - - - MIEP QP - - - -ALRAHRRRS P TS- - - -ATYGS S - - - P P ENTMF TAQARRMAKG- KLRSALQEKKRRDRLS LMS GTAAS I AKAKRRAS S AAP LP NGP GGGNGS AS TP GP AVS RP S I AKVS HRAS I GHGRS S I GGHRASF GGS LHGRAS MGAHHRAS I GAGGHP GLGGAGRRS S MGV EKKQEVNS ES TQLDQLLAED- - - S S TDDVS LP H- - - -LDTI DLDGS P VP KVP D- - - LNFS RANMDS - - PTF- - - DK- HNMNNDRS - - - - FLVP S - - - -RDARNS - - - -LTS LSLHS N- - - ELEP ELHQDQDAI A- - - -SEVDVS MQDTTFNE- - - - - - EKELENKPAVLGPR- - - -SLPI KKNTS- - - QRRTTF G- - - GDI RP RDF S F S GRDI - - - -R GGGP LAS ATNGIS RHRKGS GLEKSAAVATTRKGGIP LPTIAGSPP VRS GTTGLAARRAS LGP NS AWLVES GMMGP KS GI GLTNAQP RRMGI NGNGS GF GKGSP NGGEEDHNSAS GTMAANR - - - -ILNNEAIHNF DFSKP - - KTRQSLSSLTTLHLSNPA- - - N- - - I I RKS L- - - -SMAENEEEKA- T - - - VAKNKS SHS S KWP T- - - HTLS PI ITTALKQP VRRI S - - - -LVSQPL- - - QKTGGTENTP NA - - - -QGPSTP S AP TTAVPRRKMRSS L- - - -LTHQS LLATA- - - LSAIGGF LSGFGSFDGKNRRI TI GAGP I GKKS S S S LWQ - - - RK

KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3 KipB NCU06144.1 Klp5 Klp6 Kip3

Fig. IV.3: Alignment of A. nidulans KipB with homologous kinesin sequences from N.

crassa (NCU06144.1), S. pombe (Klp5 and Klp6) and S. cerevisiae (Kip3). The alignment was done with DNAstar using MEGALIGN (CLUSTAL) with a window size of 5 and a gap length penalty of 10. The beginning and the end of the highly conserved motor domains are indicated by asterisks above the sequences. The ATP-binding motif is boxed (orange) and the putative microtubule binding pocket is indicated by lines (green) above the sequences.

The 18 amino acid motif at the N-terminus, which is conserved among the compared proteins is highlighted by a dashed line (grey) above the sequences. The putative coiled-coil domains are marked by the diamond (blue) above the sequences.

Results

KipB 108 kDa ATP

hydrolysis

Microtubule binding

region

Coiled-coil N-terminal

18 aa.

motif

Motor domain

A

6 0 0 probability 0

2 0 0 1 .0

0 .5

a m in o a c id s

4 0 0 8 0 0 9 8 9

B

Fig. IV.4: Domains and coiled-coil prediction for the KipB protein. (A) Schematic drawing of different domains, with correspondent colour display onto the protein alignment from Fig IV.3. (B) Coiled-coil prediction (Lupas et al., 1992) for KipB (window size 14).

Significant coiled-coil probability was found between amino acids 390-408, and 500-600.

The sequence of A. nidulans kipB was deposited in the EMBL database and is available under the accession number AJ620863.

After obtaining the full length kipB gene sequence, the question which rose was if there are other kipB kinesin homologues in A. nidulans genome, as it was described for klp5 and klp6 in S. pombe (West et al., 2001). Upon completion of the A. nidulans genome sequencing project at the Whitehead Center for Genome Research (Cambridge, USA), the public databases were thus searched using the BLAST program available at this site (http://www-genome.wi.mit.edu/annotation/fungi/aspergillus/index.html) for kinesin-like proteins (Fig. IV.5) (study realized in collaboration with Sven Konzack, Marburg). The motor domain of A. nidulans KipB protein was used to identify eleven putative kinesin motors.

Results

Fig. IV.5: Relatedness analysis of the eleven A. nidulans and the ten N. crassa kinesins. A most likely phylogenetic tree of 74 kinesins was built with Treepuzzle (http://www.tree-puzzle.de/) using a maximum-likelihood algorithm. For evaluation of statistical significance of the topology 25.000 replicating puzzling steps were performed. The substitution model Whelan-Goldman 2000 was used because it produced a consensus tree, which was in good agreement with the published data (Schoch et al., 2003). For the construction of the tree we have chosen fungal kinesin sequences and additional kinesins from other organisms characteristic for the different families. It has to be noted that for A.

nidulans exon/intron boarders were only experimentally determined in the case of BimC, KlpA, KinA, KipA and KipB. In the case of N. crassa only the conventional kinesin NcKHC has been analyzed experimentally. The other primary structures of the proteins of A. nidulans and N. crassa are based on the predictions in the annotation process at the Whitehead Institute. The N. crassa Kip3 homologue was annotated manually. The origin of the kinesin sequences was abbreviated: An = A. nidulans; Nc = N. crassa; Spo = S. pombe; Ce = Caenorhabditis elegans; Mm = Mus musculus; Um = U. maydis; Sc

= S. cerevisiae; Ch = Cochliobolus heterostrophus; Hs = Homo sapiens; Ca = Candida albicans; Dm = Drosophila melanogaster; Cg = Cricetulus griseus; Sp = Strongylocentrotus purpuratus. The A.

nidulans and the N. crassa sequences were boxed.

Results

They grouped into nine of the eleven families, two kinesins being found in the Unc104 family and one did not fall into any of the known families. The ten N. crassa kinesins (identified through similar procedure of motor domain search at http://www-genome.wi.mit.edu/annotation/fungi/neurospora/index.html were closely related to the A. nidulans proteins. In comparison, in the single cell organisms S. cerevisiae reside six and in S. pombe nine kinesins (Schoch et al., 2003).