Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11172-11176, December 1993 Biology

Cytoplasmic dynein is required for normal nuclear segregation in yeast (motor protein/cytoplasmic microtubules/nuclear division/)

D. ESHEL*t, L. A. URRESTARAZU*, S. VISSERSt, J.-C. JAUNIAUX*§, J. C. VAN VLIET-REEDIJKI, R. J. PLANTA1, AND I. R. GIBBONS*

*Pacific Biomedical Research Center, University of Hawaii, Honolulu, HI 96822; tUniversite Libre de Bruxelles, Laboratoire de Physiologie Cellulaire et de Genetique des Levures, B-1050 Brussels, Belgium; *Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, D-6900 Heidelberg, Germany; and lVrije Universiteit, Biochemisch Laboratorium, Amsterdam NL 1081, The Netherlands Communicated by David J. L. Luck, July 27, 1993 (receivedfor review June 21, 1993)

ABSTRACT We have identified the gene DYNI, which (18). Synchronization of yeast cultures was achieved by encodes the heavy chain of cytoplasmic dynein in the yeast treating with a mating factor as described by Berlin et al (19). Saccharomyces cerevisiae. The predicted amino acid sequence PCR Primers. Oligonucleotide primers were designed from (Mr 471,305) reveals the presence of four P-loop motifs, as in the deduced amino acid sequence of the sea urchin dynein ,B all dyneins known so far, and has 28% overall identity to the heavy chain (20, 21) in regions that were conserved between dynein heavy chain ofDictyostelium [Koonce, M. P., Grissom, sea urchin dynein isoforms (22). The nucleotide sequences of P. M. & McIntosh, J. R. (1992) J. CeUl Biol. 119, 1597-1604] the two degenerate primers that were used to PCR-amplify with 40% identity in the putative motor domain. Disruption of pDLP1 from yeast genomic DNA are 5'-CCTGCTGGNAC- DYNI causes misalignment of the spindle relative to the bud NGGNAARAC-3' (sense strand, targeting amino acid- neck during cell division and results in abnormal distribution sequence PAGTGKT) and 5'-TACCCIGGRTTCATIGTDA- of the dividing nuclei between the mother cell and the bud. TRAA-3' (antisense strand, targeting amino acid-sequence Cytoplasmic dynein, by generating force along cytoplasmic FITMNPG). microtubules, may play an important role in the proper DNA Sequencing. Nucleotide sequencing of the original alignment of the mitotic spindle in yeast. probe, pDLP1, and other restriction fragments in the vicinity of the putative hydrolytic ATP-binding site was done by Nuclear and cytoplasmic microtubules in the yeast Saccha- subcloning into M13mpl8/mp19 vectors and using universal romyces cerevisiae participate in several well-defined cellu- M13 primers and a Sequenase 2.0 DNA sequencing kit lar processes that include the segregation of (United States Biochemical). and the migration ofthe nucleus during mitosis as well as the The nucleotide sequence of the complete gene was deter- migration and fusion of nuclei in karyogamy (1). These mined as part of the ongoing project to sequence the entire processes are believed to involve microtubule-based motor XI in yeast (strain S288C). Relevant cosmids enzymes, and recently several genes containing sequence were sonicated, and the resulting DNA fragments were regions with homology to the motor domain characteristic of repaired by using Klenow fragment and T4 DNA polymer- the kinesin superfamily have been identified in S. cerevisiae ases. Fragments ranging from 400 to 600 bp were separated and other fungi (2-8). by electrophoresis and ligated into the Sma I site of an The cytoplasmic form ofthe microtubule-associated motor M13mpl8 vector. Double-stranded were prepared dynein has ATPase activity and moves toward the minus from clones and sequenced with a Pharmacia T7 sequencing ends of microtubules in in vitro gliding assays (9). Cytoplas- kit. Gaps were filled in using specific oligonucleotide primers. mic dynein exists in a wide variety of eukaryotic cells, The entire sequence was determined on both strands. including Dictyostelium (10, 11), and indirect evidence has Fluorescence . Cells grown to mid-logarithmic suggested that it plays a major role in the movement of phase in yeast extract/peptone/dextrose (YPD) medium at chromosomes toward the minus ends of spindle microtubules 30°C were fixed with 4% formaldehyde and viewed by at the anaphase stage of mitosis (12, 13), as well as in the fluorescence microscopy after microtubules with a retrograde transport of organelles in nerve axons (14-16). monoclonal against ,B- (YOL1/34) and stain- However, direct evidence for the in vivo functions of cyto- ing DNA with 4',6-diamino-2-phenylindole (1 pg/ml) by plasmic dynein has been lacking. standard techniques (17). We have now identified and sequenced the gene, DYNI, Assay for Chromosome Loss. The method used for assess- which encodes the heavy chain of cytoplasmic dynein in the ing elevated levels of chromosome loss was based on that yeast S. cerevisiae.ll Disruption of the DYNI gene causes described by Berlin et al. (19) to assay loss of chromosomes misalignment of the spindle relative to the bud neck during in bikl mutants. Four colonies of wild-type and mutant cell division and results in an abnormal distribution of the diploid strains (DEY314: a/a ade2 canl+ his7/+ leu2/+ nuclei between mother and trpl ura3 ade5/+ cyh2/+ lys5/+ trp5/+ leul/+ his3/+ dividing daughter cells. lys2/+ and DEY 316: a/a ade2 canl/+ his7/+ leu2/+ trpl ura3 ade5/+ cyh2/+ lysS/+ trpS/+ leul/+ his3/+ lys2l+ MATERIALS AND METHODS dyni-A:: URA3) were inoculated into YPD medium and Media and Microbial Techniques. Yeast media and genetic grown at 30°C to a density of107 cells per ml. Washed aliquots techniques were essentially as described in Rose et al. (17). of the cultures were then plated on YPD plates to test for Transformations were done with the lithium acetate method tTo whom reprint requests should be addressed at his present address: Department of Biology, Brooklyn College, 2900 Bedford The publication costs of this article were defrayed in part by page charge Avenue, Brooklyn, NY 11210. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. Z21877). 11172 Downloaded by guest on September 24, 2021 CeH Biology: Eshel et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11173 viability and on YPD plates that contained cycloheximide at consensus motif, have yielded no evidence for a second 10 mg/ml (Sigma) to assess the frequency of appearance of dynein gene. colonies resistant to cycloheximide. To distinguish between pDLP1 was used to identify overlapping cosmids contain- resistance due to loss of chromosome VII and that due to ing DNA inserts that cover DYNI, which were distributed mitotic recombination, cell aliquots were also plated on among several laboratories as part of the project sequencing medium that lacked leucine. Colonies that became resistant the entire chromosome XI. The nucleotide sequence of to cycloheximide due to chromosome loss were leucine DYNI showed a continuous open reading frame of 12,276 bp auxotrophs, whereas those that became resistant due to with no consensus motifs for introns. The deduced amino mitotic recombination in the 80-centimorgan LEUI-CYH2 acid sequence (4092 residues, Mr 471,305) is presented in Fig. interval were leucine prototrophs. 1, and a restriction map of DYNI is shown in Fig. 2. Four P-loop motifs, GXXXXGKT, characteristic of most ATP- binding sites (24) are present in the mid-region of the heavy RESULTS chain, at positions 1796, 2074, 2418, and 2760 (Fig. 1 and Identification and Characterization of DYNI. Using degen- P1-P4 in Fig. 2), which are similar to those in dynein heavy erate primers (see Materials and Methods), we PCR- chains of Dictyostelium and sea urchin (11, 20, 21). The amplified from genomic DNA of S. cerevisiae a 305-bp DNA amino acid sequence of DYNI shows 28% identity when fragment that was cloned and named pDLP1 (for dynein-like aligned with the sequence ofcytoplasmic dynein heavy chain protein). The predicted amino acid sequence encoded by from Dictyostelium (11) and 19%o identity to the axonemal (3 pDLP1 has 47% identity to that of the corresponding region heavy chain from sea urchin (20). In the putative motor of the sea urchin P heavy chain (20) with a perfect match in domain that contains the four P-loops (residues 1780-2800) the CFDEFNR region, which is absolutely conserved in all this amino acid sequence is 40% identical to that of Dictyo- stelium. dynein sequences known so far (11, 20-23). Disruption ofDYNI. An 11-kb DNA clone containing =7 kb On a yeast chromosome blot, pDLP1 hybridizes solely to of dynein coding sequence was isolated by screening a yeast chromosome XI. On a mapping blot supplied by Maynard genomic library (25) with pDLP1. Two EcoRJ restriction Olson (Washington University School of Medicine, St. Lou- fragments from this clone were chosen for the disruption is), pDLP1 hybridized to a single clone located =100 kb to the experiments because they contained internal HindIII sites right ofthe centromere on chromosome XI between the genes that permit deletion of dynein coding sequences and substi- TIFI and GAP]. On a Northern blot of yeast poly(A)+ RNA, tution by a 1.1-kb HindIII fragment containing the yeast pDLP1 hybridized to an -13-kb band, consistent with the URA3 gene. One is a 3.5-kb fragment, R2, coding for a region characteristically large size of dynein heavy chains in other containing the putative hydrolytic ATP-binding site and the organisms (11, 20-23); this gene was named DYNI. other, R4, is a 0.9-kb fragment closer to the 5'-end ofthe gene Attempts to find other dynein heavy chain genes in S. (Fig. 2). The disrupted R2 or R4 fragments were used to cerevisiae, either by probing Southern blots of the genomic transform a haploid and a diploid Ura- strain (YNN281, a DNA with pDLP1 at low stringency or by PCR amplification, trpl-A his3-A200 ura3-52 lys2-801a ade2-10 gal mal CUpr and using a different degenerate primer based on the CFDEFNR strain DEY310, a/a trpl-A his3-A200 ura3-52 lys2-801a 1 0 20 30 40 50 60 70 so 90 1 00 1 10 1 20 1 30 MCKNEARLANELIEFVAATVTGIKNSPKENEQAFIDLuHLt,YLERFQFFLGLLDGREFDTLFVFLFEELMRTI v I IuGEEAIYDANLANKKYSTLLI IKSRSVIvuAtrILATQISAIYLPGPVNAGNLA 131 SIITHGVSSVFGQLIKSDTKTYSVETIDKTRRKLDWISKQFQQLHTSIETPDLLAMVPSI IKLAVSKGATSHDYANYLPSNDLESURFLNILQSIANKWVFLVLKQTLAIDRDIKtFLDEVEFWSNFYE 261 VLKSLIEQTQSQEFQVCLSVLTNAKRFHNLTNLLNEGSLSDKFKLADKYNQFLSSIPIDEVRQASNLEDLQELFPVLASSLKKFRYSGYPVQRFVVLMDKISOEVMDAILSNLSDLFQLEYGSFLGLYEK 391 SAGMIEEWDDIVQDVNLL IREDLRKRAPOELLIQKLTFTSASVKATLDEI LSTRKRFFSLAETIKSISPSTYHEEIQRLYHPFEQIHDISVNFRLKLEQAESEFSKNVLDLEKKLQNTLASFMDSDHCPT 521 EKLSYLVKFKPLMELCPRIKVKVLENQQI LLLEIKKDIROLETGLELLPKILHVEALNNIPPISARISYFLNVQSRIDNIVQYLEALFGSNWNDTLEGRSISTSIVQLRKETNPHDVFLHWLGNFPEKAT 651 ANLLTTPI LKLIRNNEDDYELKVNFDFALAAAYSELRSLTYMAQVPSHIVRIARTYMYLYPRAINLVELIQTFFSLSKSLSYTFYTNIFLKRNVQTVWLLLQQILITPWESLQEESSEMSCSVHSLARL 781 EKSIDGILSDYQILKNSEPQFAKEFSGLKSFDGTADDLHEVEEI ISNIQAIFENLFTKGLTNVSDHISTFNNLI ISIILEKVRLNLKKMHFPKHVLKLSFNEGRITSSPSLAAKRSLLKDIEALLNKVV 911 LINFLHDPDHPLSTTLTFNSLVIKLKDDIQNCIEQVQNLHCKINSYKWIKMEFLWQITEEAFLEVVDNSTORCFGILKGLLDSQSKFDLI ISRNNFSKNLVLHTEDAQRHIRSKMDSWILYVSKHLLT 1041 IYERDARKLHEDMNRDREAVEDMDINFTSLKNITVI IEAVNVNKRHLTERDIQIKLLGSVMRLTKLKVRFPSHFVYIDQLDNDFSSLROSLSYVEQELQKHRVVIAKSLEEGVENINNLSOSLNESWSV 1171 RKPISPTLTPPEALKILEFFNESITKLKKKWIISVAAAAKMLLIPVVLNDQLTHVVEEVKTYDLVWRSIKNLWEDVORTFETPWCRVDVLLLQSWLANFLRRADELPRAVKQFEMYKSLFSQVNMLTSVNK 1301 ILVELKDGALKPRHWNMIFRDIGKRQIQKNLLDKLEFSLKDVMVLNLTLNEI LLTKI IERAQKEFVIEKSLNRIKFEAYEVIEHSSGLKLVREWDVLEQACKEDLEELVSMKSNYYKI FEQDCLO 1431 LESKLTKLSEIQVNWVEVQFYWLDLYGI LGENLDIQNFLPLETSKFKSLTSEYKMITTRAFOLDTTIEVIHIPNFDTTLKLTIDSLKMIKSSLSTFLERQRRQFPRFYFLGNDDLLKI IGSGKHHDQVSK 1561 FMKKMFGSIESI IFLEDFITGVRSVEGEVLNLNEKIELKDSIQAQEWLNI LDTEIKLSVFTOFRDCLGQLKDGTDIEVVVSKYI FQAILLSAQV - TELVEKCLQTNQFSKYWKEVDVKIKGLLDKLNKS 1691 SDNVKKKIEALLVEYLHFNNVIGQLKNCSTKEEARLLWAKVQKFYOKNDTLDDLNSVFISQSGYLLQYKFEYIGIPERL IYTPLLLIGFATLTDSLHQKYGGCFFGPAGTGKTETVKAFGQNLGRVVVVF 1821 NCDDSFDYQVLSRLLVGITQIGAWGCFDEFNRLDEKVLSAVSANIOQIQNGLQVGKSHITLLEEETPLSPHTAVFITLNPGYNGRSELPENLKKSFREFSMKSPQSGTIAEVI LQIMOFEDSKSLASKIV 1951 HFLELLSSKCSSMNHYHFGLRTLKGVLRNCSPLISEFGEGEKTVVESLKRVI LPSLGDTDELVFKDELSKI FDSAGTPLNSKAIVQCLKDAGQRSGFSMSEEFLKKCMQFYYM KTQQALILVGKAGCGK 2081 TATWKTVIDAMAIFDGHANVVYIDTKVLTKESLYGSIVKATLEWRDGLFTSI LRRVNDDITGTFKNSRIWVVFDSDLDPEYVEAMNSVLDDNKI LTLPNGERLPIPPNFRILFETDNLDHTTPATITRC 2211 GLLW'FSTDVCSISSKIDHLLNKSYEALDNKLSMFELDKLKDLISDSFDMASLTNI FTCSNDLVHILGVRTFNKLETAVQLAVHLISSYROWFQNLWOKSLKDVITLLIKRSLLYALAGDSTGESO AFIQ 2341 TINTYFGHDSQELSDYSTIVIANDKLSFSSFCSEIPSVSLEAHiEVMRPDIVIPTIDTIKHEKI FYDLLNSKRGI ILCPPGSGKTMI"NNALRNSSLYDVVGINFSKDMEHI LSALHFRHTNYVTTSKG 2471 LTLLPKSDIKNLVLFCDEINLPKLDKYGSONVVLFLRQLMEKQGFWTPENKWVIERIHIVGACNPPTDPGRIPMSERFTRHAA LYLGYPSGKSLSQIYEIYYKAI FKLVPEFRSYTEPFARASVHLY 2601 NECKARYSTGLOSHYLFSPRELTRLVRGVYTAINTGPROTLRSLIRLWAYEAWRI FADRLVGVKEKNSFEQLLYETVDKYLPNQDLGNISSTSLLFSGLLSLDFKEVNKTDLVNFIEERFKTFCDEELEV 2731 PMVIHESMVDHI LRIDRALKQVQGHML IGASRTKILTRFVAWLNGLKIVQPKIHRHSNLSDFDMI LKKAISDCSLKESRTCLI IDESNILETAFLERMNTLLANADIPDLFQGEEYDKLLNNLRNKT 2861 RSLGLLLOTEQELYDWFVGEIAKNLHVVFTICDPTNNKSSAMISSPALFNRCI INWMlGODMTKrMSOVANNMVDVIPMEFTDFIVPEVNKELVFTEPIQTIRDAVVNI LIHFDRNFYQKVKVGVNPRSPG 2991 YFIDGLRALVKLVTAKYQDLQEWIRFVNVGLEKLNESVLKVNELNKTLSKKSTELTEKEKEARSTLDKMLMEONESERKOEATEEIKKI LKVQEEDIRKRKEVWVSIQDIEPTILEAfiRGVKNIKKQQL 3121 TEIRSMVNPPSGVKIVMEAVCAILGYQFSNWRDIQQFIRKDDFIHNIVHYDTTLHMKPIRKYMEEEFLSDPNFTYETINRASKACGPLYQWVNAGINFSKVLENVDPLRQEMKRIEFESLKTKANLLAA 3251 EEMTODLEASIEVSKRKYSLL IRDVEAIKTEMSNV - NLDRSISLVKSLTFEKERWLNTTKOFSKTSOELIGNCI ISSIYETYFGHLNERERADMLVILKRLLGKFAVKYDVNYRFIDYLVTLDEKMNL 3381 ECGLDKNDYFLENVSIVMNSQDAVPFLLDPSSHMITVISNYYGNKTVLLSFLEEGFVKRLENAIRFGSVI IQDGEFFDPIISRLISREFNHAGNRVTVEIGDHEVDVSGDFKLFIHSCDPSGDIPI FLR 3511 SRVRLVHFVTNKESIETRI FDITLTEENAEMORKREDL IKLNTEYKLKLKNLEKRLLEELNNSQGNULENDELMVTLNNLKKEAMNIEKKLSESEEFFPQFDNLVEEYSI IGKHSVKI FSMLEKFGQFHW 3641 FYGISIGQFLSCFKRVFIKKSRETRAARTRVDEI LWLLYGEVYCQFSTALDKKFKMIMATMFCLYKFDIESEQYKEAVLTMIGVLSESSDGVPKLTVDTNNDLRYLWDYVTTKSYISALNWFKNEFFVD 3771 EWNIADVVANSENNYFTMIASERDVDGTFKLIELAKASKESLKI IPLGSIENLNYAQEEISKSKIEGGWI LLONIQVSLSWVKTYLHKHVEETKAAEEHEKFKMFMTCHLTGDKLPAPLLORTDFtFVYEDI 3901 PGI LDTVKDLWGSQFFTGKISGVWSVYCTFL LSWFHALITARTRLVPHGFSKKYYFNDCDFQFASVYLENVLATNSTNNIPWAQVRDHIATIVYGGKIDEEKDLEVVAKLCAHVFCGSDNLQIVPGVRIP 4031 OPLLOOSEEEERARLTAI LSNTIEPADSLSSWLQLPRESI LNYERLOAEVASSTEQLLQEM FIG. 1. Deduced amino acid sequence of the cytoplasmic dynein heavy chain in S. cerevisiae. The four P-loop motifs are underlined at positions 17%, 2074, 2418, and 2760. Downloaded by guest on September 24, 2021 11174 Cell Biology: Eshel et al. Proc. Natl. Acad. Sci. USA 90 (1993) R4 R2 I I R G R SH H R HHH S R R 5,

RR R HG H RBRG G R P2- I P31 P4I 111 1 1 3'

FIG. 2. Restriction enzyme map ofDYNI. R, EcoRI; H, HindlIl; S, Sac I; G, Bgl II; and B, BamHI. Locations of the four P-loops (P1-P4) and of pDLP1 are indicated. The DNA restriction fragments R2 and R4 were disrupted with a copy of the yeast URA3 gene at the positions indicated and were then used to transform the cells. ade2-10 gal mal CUPr) by integrative transformation. Colo- cytoplasmic microtubules. In -3% of the mutant cells with nies of transformants from both haploid and diploid strains abnormal nuclear segregation, the spindle contained within that appeared on the selective plates indicated that the the mother cell was longer than the cell diameter and bent disruption of dynein is not lethal for vegetative growth. (Fig. 3E). Dissection of tetrads from the sporulated diploid transform- Table 1 shows the distribution of nuclear morphologies in ants revealed spore viability and segregation of URA3, which dynl cultures that were synchronized by a release from G, agreed with the above conclusion. Southern blot analysis of arrest, induced by a mating factor. After correction for the EcoRI-digested DNA from the transformants verified that the binucleated cells that were carried over from previous divi- URA3 insertions had occurred at the expected loci in both sions before the arrest, the data suggest that in the first cell haploid and diploid strains. We chose to further characterize cycle, 2 hr after the release, 38% ofthe total complete nuclear the mutants that were transformed with the R2 disrupted divisions showed improper segregation ofthe nuclei between fragment. mother and bud. Examination at later stages appeared to The Phenotype of Dynein-Disrupted Cells. dynl -A:: URA3 show a reduced percentage ofbinucleated cells, although the haploid cells grew at a similar rate to the wild-type cells, over diminished synchrony makes quantification unreliable. the temperature range of 14-37°C in rich liquid medium. dynl The possible role of cytoplasmic dynein in chromosome and wild-type strains of opposite mating types were crossed segregation (12, 13) was assessed by determining the rate at in different combinations. The viability of the tetrads result- which one copy ofchromosome VII became lost from a dynl ing from sporulation of heterozygous or homozygous dyni x dynl diploid strain. Such colonies appeared at a frequency diploids appeared normal, suggesting that cytoplasmic dy- of -3 x 10-6 in both the wild-type (strain DEY314) and the nein was not essential for meiosis or karyogamy. mutant diploids (strain DEY316). These results, which show DNA staining of nuclei in the mutant strain showed sig- no indication of elevated chromosome loss in dynl x dynl nificant aberrations in nuclear segregation (Fig. 3). The cells, suggest that dynein does not play a critical role in typical budded wild-type cell (Fig. 3A) has two nuclei fully chromosome segregation in yeast, although a supportive or divided and segregated between the mother cell and the bud. redundant role remains possible. The nuclear microtubules that contain the spindle are aligned with the axis of the neck. In those wild-type cells that have not yet completed their nuclear division, the spindle is always DISCUSSION oriented parallel with the neck axis. In the dynl mutant The Cellular Roles of Cytoplasmic Dynein. In this work we strain, on the other hand, =25% of the budded cells had a have identified and characterized the DYN1 gene, which large binucleated mother and an anucleated bud (Fig. 3 B and encodes a cytoplasmic dynein heavy chain in the yeast S. C). The spindle in these cells does not penetrate the neck, and cerevisiae. The disruption ofthis gene suggests that dynein is it is aligned with the axis ofnuclear division, in an orientation responsible for alignment ofthe mitotic spindle with the neck that seems random relative to the neck axis. For example, the axis, a process required for the fidelity ofnuclear segregation spindle in Fig. 3B is approximately perpendicular to the neck during mitosis. However, dynein is not essential for vegeta- axis, whereas the spindle in 3C is parallel with it. Cytoplasmic tive growth. microtubules and spindle pole bodies are clearly visible in the The dynl phenotype generally resembles that of the tem- dynein mutants. Approximately 2% of the dynl-containing perature-sensitive in the yeast /tubulin gene, tub2- cells accumulate more than two nuclei in the mother cell (Fig. 401 (26). This similarity suggests that normal nuclear segre- 3D), which accommodates a complex array of nuclear and gation may be a result of the interaction of dynein with the Table 1. Nuclear distribution in cells of synchronous cultures Nuclear morphology in budding cells that Cells counted,* no. completed nuclear division MATa ura3 DYNI MATa ura3 dynl-A::URA3 One nucleus in mother/one nucleus in bud 211 245 Two nuclei in mother/none in bud 1 (0.5%)t 336 (38%)14 Cells were fixed before all nuclear divisions were completed to assure that only the first generation of division was monitored. Approximately 5o of budding cells in wild-type and dynl cultures had completed nuclear division; these calculations are based on this subpopulation. *Numbers represent sum from three independent experiments. tPercentage of binucleated mother cells with anucleated buds out of total nuclear divisions. tPercentage is corrected for number of binucleated cells carried over from previous generations as reflected in arrested cultures, where 0 and 20% of wild-type and dynl cells (respectively) were binucleated or anucleated. Downloaded by guest on September 24, 2021 Cell Biology: Eshel et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11175

cytoplasmic rnlicrotUl lcIICs cytoplasr mic clynein A

. :'I LJcI0,a .TIcrotL ibLles spinidle pole bDody

B r UcIea r orlvelole

FIG. 4. Model for the role of cytoplasmic dynein in aligning the spindle and pulling it toward the neck. The thin arrow shows the direction in which dynein "walks" along the microtubules, and the C thick arrow indicates the resultant spindle movement. I... , dent forces. One force may operate from within the spindle to elongate it by sliding antiparallel nuclear microtubules with akinesin-like motor protein (6-8), whereas the otherpulls the spindle pole bodies apart by dynein exerting force on cyto- plasmic microtubules (Fig. 4). The similar overall growth rates of the dynl and wild-type cells may be accounted for by a recovery mechanism within the cell that results in one of the nuclei in the binucleated mother cells finding its way to the neck and entering the bud late but before . Such a mechanism may be more likely to succeed when the two sister nuclei are distributed parallel to the neck axis (Fig. 3C) rather than perpendicular to it (Fig. 3B). In addition, binucleated cells that further divide may have a higher probability oftransferring a nucleus to the bud in the next . A recovery hypothesis is supported by the preliminary finding of a reduced percentage of binucleated cells at later stages after the release from arrest. A similar explanation was proposed for bikl cells by E Berlin et al. (19) to explain the discrepancy between the growth rate of the mutants and their aberrant nuclear mor- phologies. The immunolocalization of cytoplasmic dynein to mitotic kinetochores in higher cells (12, 13) has suggested that dynein phase DAPI tu bu Inplays a role in chromosome movement toward the poles. However, our present results that show no indication for an FIG. 3. Bright-field images and fluorescent stainiing of DNA and elevated level of chromosome loss in homozygous dyni microtubules of control cell (A) and dynl cells (B-E) from asynchro- diploids suggest that if cytoplasmic dynein plays a role in nous logarithmic-phase cultures (strain YNN281 D)OYNI and strain chromosome movement and segregation in yeast, this role is

DEY306 dyn)::URA3, respectively). (Bar = 10 PM.') probably functionally redundant with at least one other motor

astral microtubules that are thought to be Ilacking in the protein. A similar redundancy is believed to occur with the tub2-401 mutants at their restrictive temperatiture. A simple pair of kinesin-like proteins Cin8 and Kipl (7) and with the model would suggest that dynein binds to the binner plasma pair of the kinesin-like protein Smyl and the yeast myosin membrane in the vicinity of the neck, eith4er erdirectlydrecty or heavy-chain Myo2 (3). Amino Acid Sequence Comparison with Other Dyneins. through an accessory structure. By walking a]long the cyto- Sequence comparison of the P-loops in the cytoplasmic

plasmic microtubules toward their minus ends,, dynein could dyneins from yeast, Dictyostelium, and sea urchin shows that pull the spindle and related structures to the inieck and align the characteristic ATP-binding motif GPAGTGKT at P1, them with the neck axis (Fig. 4). The long bent spindle which is probably the hydrolytic site (20), and the corre- appearing in some of the dynl cells (Fig. 3E),,vwhich has also sponding motif GPPGSGKT at P3 are conserved not only been reported in the tub2-401 mutants (26), sugggests that the among the three organisms but also between these two forces within the spindle keep elongating it during anaphase, P-loops (Fig. 5). The P-loop motifs at P2 and P4 are, in but because the spindle is misaligned with respeect to the neck general, less conserved (data not shown). Analysis of the and the spindle pole bodies cannot be pulled furither apart, the sequences in two regions downstream from P1 and P3 re- microtubules constituting the spindle cannot e-xtend beyond vealed patterns that are relatively conserved in all three the diameter of the mother cell, and they bec4 ome bent. organisms (Fig. 5). The completely conserved CFDEFNR More generally, these results suggest that sj pindle elonga- motif that is located %50 residues downstream from P1 in all tion at anaphase B is accomplished by at least two indepen- dyneins known so far (11, 20-23) shows substantial homology Downloaded by guest on September 24, 2021 11176 Cell Biology: Eshel et al. Proc. Natl. Acad. Sci. USA 90 (1993)

SU p (1852) 1 G P A G T G K T E TT K D L G R A L G I protein functions in yeast (3, 6, 7). The potential interplay of ~~:: s ::: dynein with other motor proteins will provide scope for much Dicty (1969) 1 G P A TG K T E T V K A L G S QL G R additional study. P1 SU cy (?) 1 G P A G T G K T E S V KA L G H QL G R LYeast (1796) 1 G P A GT GK T E T V K A F GQ NL G R We thank the following for their help and advice: I. Becker, M. V. Dicty (2669) 1 G P P G S G K T M T L T S T L R A F PD Borre, B. Dujon, B. H. Gibbons, C. Jallet, C. Jauniaux, M. V. P3 SU cy (?) 1 G P PG S G K T M T L FS A L R A L P D GK Olson, C. A. Phillipson, H. Ren, L. Riles, M. D. Rose, J. F. Scott, Yeast (2418) 1 [G P PLG S TIM I M N N A L R N S S L and W.-J. Y. Tang. This work was supported by grants from the 1 G N A G V National Science Foundation, the National Institute of General LSU P (2460) G L K S L V G D K L S N L G E Medical Sciences, the European Communities Commission program Biotechnology Research for Innovation, Development, and Growth 43 L S QT G A W G C - F - D E F N R I S V E V L S VV * * in Europe, the Region de Bruxelles Capitale and by grant 2.4547.92 *C[ *[ *G--D -ERLEERILSAV 43 L C Q GCG AWGC-F_ DEFNRLEERILSAV from the Fonds de la Recherche Fondamentale Collective. Pi 43 L C QV G A W G C - F - D E F N R L E E R M L S A V _43 I T QI G A W G C - F - D E F N R L D E K V L S A V 1. Huffaker, T. C., Thomas, J. H. & Botstein, D. (1988) J. Cell Biol. 106, 1997-2010. 59 P T QL G K W L VV F C DE I N - L P S T D K Y G T P3 58 P V Q L G]K W L VV F C D E I N - L P D M D Q Y G T 2. Meluh, P. B. & Rose, M. D. (1990) Cell 60, 1029-1041. 58 P K S D I KLjFGC1DWN L V E ILN _LL PLK L D K Y G S 3. Lillie, S. H. & Brown, S. S. (1992) Nature (London) 356, 358-361. 56 P - P G TK KL V YF I DD M N - M P E V D T Y G T 4. Enos, A. P. & Mornis, N. R. (1990) Cell 60, 1019-1027. 5. Hagan, I. & Yanagida, M. (1990) Nature (London) 347, 563- 93 L I P S V G I F I - T M N P G Y A - G R T E L P E N 566. 6. Roof, D. M., Meluh, P. B. & Rose, M. D. (1992) J. Cell Biol. 94 L H Q DI M G I F v- T }1 N P G Y A - GR SN TiP D N 118, 95-108. P1 101 v N P DJMA IFI- T M N P G Y A - GR SN|L|P D N 7. Hoyt, M. A., He, L., Loo, K. K. & Saunders, W. S. (1992) J. 93 L S P H T A V F|I - TL N P G Y N - G R S E|L|P E N Cell Biol. 118, 109-120. 109 I K L jDK I Q F V G A C N P P T D A G R V Q L T H R 8. Saunders, W. S. & Hoyt, M. A. (1992) Cell 70, 451-458. P3 108 V R FE R I Q F V|G A C N P P T D PG R K PLbS HR 9. Vallee, R. B. & Shpetner, H. S. (1990) Annu. Rev. Biochem. 108 V T I E R I HIV G A CN P P TD PIG R I P M S E R 59, 909-932. 10. Skoufias, D. A. & Scholey, J. M. (1993) Curr. Opinion Cell 104 K E I H K C Q Y V S C M N P - T - A G S F T I N S R Biol. 5, 95-104. deduced amino acid sequences in 11. Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. FIG. 5. Alignment of selected Cell Biol. 119, 1597-1604. heavy chains of cytoplasmic dynein from yeast, Dictyostelium 12. Pfarr, C. M., Coue, M., Grissom, P. M., Hays, T. S., Porter, (Dicty) (11), and sea urchin (SU-cy) (22). Regions shown are from the M. E. & McIntosh, J. R. (1990) Nature (London) 345, 263-266. vicinity ofthe first and the third P-loops (see text). Identical residues 13. Steuer, E. R., Wordeman, L., Schroer, T. A. & Sheetz, M. P. among cytoplasmic dyneins are enclosed in boxes. Residues that are (1990) Nature (London) 345, 266-268. identical between the cytoplasmic dyneins and the axonemal 14. Lacey, M. L. & Haimo, L. T. (1992) J. Biol. Chem. 267, dynein of sea urchin cilia (SU-(3) are indicated with colons. Numbers 4793-4798. indicate position relative to the first glycine of each P-loop motif. 15. Schnapp, B. J. & Reese, T. S. (1989) Proc. Natl. Acad. Sci. Numbers in parentheses are the absolute positions. USA 86, 1548-1552. 16. Schroer, T. A., Steuer, E. R. & Sheetz, M. P. (1989) Cell 56, to a similarly conserved region downstream from P3 (Fig. 5). 937-946. Homology is also observed in a second conserved region 17. Rose, M. D., Winston, F. & Hieter, P. (1990) Methods in Yeast located =100 residues downstream from both P1 and P3. Genetics: A Laboratory Course Manual (Cold Spring Harbor This sequence analysis suggests that the P1 and P3 regions Lab. Press, Plainview, NY), pp. 177-186. and functions in the native 18. Ito, H., Fukada, Y., Murata, K. & Kimura, A. (1983) J. have similar conformations heavy Bacteriol. 153, 163-168. chain, but as yet there exists only evidence for the putative 19. Berlin, V., Styles, C. A. & Fink, G. R. (1990) J. Cell Biol. 111, hydrolytic function ofthe P1 site from its proximity to the site 2573-2586. ofvanadate-mediated photocleavage on the heavy chain (20). 20. Gibbons, I. R., Gibbons, B. H., Mocz, G. & Asai, D. J. (1991) By analogy to kinesin (27), the conserved region -100 Nature (London) 352, 640-643. residues downstream from P1 has been proposed as part of 21. Ogawa, K. (1991) Nature (London) 352, 643-645. domain in 22. Gibbons, I. R., Asai, D. J., Tang, W.-J. Y. & Gibbons, B. H. the ATP-sensitive microtubule-binding dyneins (1992) Biol. Cell 76, 303-309. (28). 23. Mikami, A., Paschal, B. M., Mazumdar, M. & Vallee, R. B. Condusion. Our results suggest that cytoplasmic dynein, in (1993) Neuron 10, 787-7%. conjunction with cytoplasmic microtubules, is responsible 24. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. for the migration of the mitotic spindle to the neck and its (1982) EMBO J. 1, 945-951. alignment with the neck axis, which are required for the 25. Rose, M. D., Novick, P., Thomas, J. H., Botstein, D. & Fink, At first it G. R. (1987) Gene 60, 237-243. fidelity of nuclear segregation. sight, appears 26. Sullivan, D. S. & Huffaker, T. C. (1992) J. Cell Biol. 119, surprising that such a large and complex motor protein as 379-388. dynein should be present in S. cerevisiae when it is not 27. Yang, J. T., Laymon, R. A. & Goldstein, L. S. B. (1989) Cell completely essential for cell growth, but this hypothesis is 56, 879-889. consistent with other evidence for redundance in motor 28. Witman, G. B. (1992) Curr. Opinion Cell Biol. 4, 74-79. Downloaded by guest on September 24, 2021