Cell, Vol.

60,

1029-1041,

March 23,

1990,

Copyright 0

1990 by Cell Press

KAR3, a Kinesin-Related Gene Required for Yeast Nuclear Fusion Pamela 6. Meluh and Mark D. Rose Department of Biology Lewis Thomas Laboratory Princeton University Princeton, New Jersey 08544-1014

Summary The KAR3 gene is essential for yeast nuclear fusion during mating, and its expression is strongly induced by a factor. The predicted KAR3 protein sequence contains two globular domains separated by an a-helical coiled coil. The carboxy-terminal domain is homologous to the amino-terminal mechanochemnicel domain of Drosophila kinesin heavy chain. Mutation of the putative ATP binding site produces a dominant ‘poison” of nuclear fusion. The mutant protein shows enhanced microtubule association in viva, as predicted for a kinesin-like protein in a state of rigor binding. Localixation of hybrid proteins to cytopiasmic microtubules in shmoos indicates that the amino-terminal domain ai& contains determinants for microtubule association. Thus, KAR3 is a member of a larger family of kinesin-lika proteins characterized by the presence of the mechanochemical domain tethered to different pmtein binding domains. The phenotypes of kar3 mutants suggest that the protein mediates micmtubule sliding during nuclear fusion and possibly mitosis. introduction The yeast Saccharomyces cerevisiae is an excellent organism for the genetic analysis of the function of microtubules and microtubule organizing centers (Huffaker et al., 1987). In addition to essential functions in mitosis (Neff et al., 1983), microtubules are required for nuclear movement and nuclear fusion (Huffaker et al., 1988). In yeast, the microtubule organizing center (spindle pole body, or SPB) is embedded within the nuclear envelope and nucleates both intranuclear and cytoplasmic microtubules. The nuclear envelope remains intact throughout all stages of mitosis, meiosis, and conjugation (Byers, 1981). To form a stable diploid nucleus during conjugation, two haploid nuclei must fuse in a process known as karyogamy. Cytological observation of mating cells has implicated the cytoplasmic microtubules in karyogamy (Byers and Goetsch, 1974,1975). In early zygotes, microtubules extend from the SPBs and interconnect the two haploid nuclei. After moving together, nuclei fuse along one edge of the SPB. The requirement for microtubules was confirmed by the observation that nuclear fusion is inhibited by benomyl, a microtubule depolymerizing agent (Delgado and Conde, 1984). In addition, conditional mutations in the gene for P-tubulin, particularly alleles that affect the cytoplasmic microtubules, cause a karyogamy defect (Huffaker et al., 1988). Nuclei acquire the ability to undergo karyogamy as one

of the mating-specific responses elicited by the reciprocal exchange of peptide mating pheromones (Rose et al., 1988). Other pheromone responses (recently reviewed in Cross et al., 1988) include the transcriptional induction of several genes involved in mating, production of sexspecific agglutinins, transient arrest in the Gl phase of the cell cycle, and the formation of a rounded cell projection (called “shmooing”). With the aim of identifying proteins that interact with or regulate the cytoplasmic microtubules, we have used nuclear fusion as a sensitive assay for microtubule function. Mutations in three genes (KAR7, KARS, and KAR3) dramatically reduce the efficiency of karyogamy without affecting the frequency of cell fusion (Conde and Fink, 1978; Polaina and Conde, 1982). Nuclear fusion occurs at l%-10% of the wild-type frequency in crosses between Kar- and wild-type strains. The KAR7 gene has been implicated in SPB duplication (Rose and Fink, 1987). The KARP gene encodes the yeast homolog of mammalian BiP/GRP78, the HSP70 protein that resides in the endoplasmic reticulum (Rose et al., 1989; Normington et al., 1989). The precise roles of these two genes in karyogamy have not yet been elucidated. One function expected to be required for karyogamy is nuclear movement mediated by one or more microtubulebased mechanochemical proteins. Two proteins that generate movement along polymerized microtubules in vitro have been identified (Vale et al., 1985; Paschal et al., 1987). Kinesin supports movement toward the “plus” ends of microtubules (Vale et al., 1985; Porter et al., 1987), whereas cytoplasmic dynein supports movement toward the microtubule “minus” end (Paschal and Vallee, 1987). In addition, dynamin has recently been described as a protein that bundles microtubules and apparently generates ATP-dependent sliding between them (Shpetner and Vallee, 1989). None of these proteins has yet been isolated from yeast. Kinesin was first identified in squid axoplasm as a protein that promotes the movement of vesicular organelles along microtubules (Vale et al., 1985). Kinesin-like molecules have since been isolated from other sources, including mammalian neuronal tissues (Vale et al., 1985; Bloom et al., 1988), sea urchin eggs (Scholey et al., 1985), Dictyostelium discoideum (McCaffrey and Vale, 1989), and Drosophila melanogaster (Saxton et al., 1988). Native kinesin is a multimeric protein complex of two 120 kd heavy chains and two 82 kd light chains (Kuznetsov et al., 1988; Bloom et al., 1988). Based on DNA sequence and microtubule binding analyses, the heavy chain of Drosophila kinesin is composed of three structural domains (Yang et al., 1989). The large globular amino-terminal domain is thought to be responsible for the motor activity of kinesin because it hydrolyzes ATP, binds microtubules in vitro, and contains a consensus ATP binding site (Scholey et al., 1989; Yang et al., 1989). The central region is predicted to fold into an a-helical coiled coil, presumably mediating heavy chain dimerization. Finally, the small

Cell 1030

Table

1. Nuclear

Parental

Fusion

Phenotypes

of Various

Mutants

Strains

MA Ta MS10 MS147 MS147 MS10 MS204 MS204

KAR3

KARB KAR3 KAR3 KAR3 kar3-lOO::LEU2 kar3-700::LEU2

MATa

% Diploids

MS296 MY1124 MY1256 MS571 MS296 MS571

40 1.3 16 56 64 1020 to determine the number of as having a haploid nucleus (C/D) is inversely related to

analysis reveals that the KAR3 protein contains a large globular carboxy-terminal domain homologous to the microtubule motor domain of kinesin heavy chain. In addition, the localization of KAR3-/acZ hybrid gene products indicates that KAR3 contains a second microtubule association domain distinct from the kinesin-like domain. We propose that KAR3 is a member of a larger protein family characterized by a kinesin-like motor domain tethered to an “adapter” domain. We suggest that in vivo the KAR3 protein functions as a mechanochemical motor to effect the sliding of antiparallel microtubules. The phenotypes of various kar3 mutants imply that this function of the protein is essential for nuclear fusion and important during mitosis. Results

+5.0 KD Hydra

GOR Turns GOR Alpha GOR Beta

CID

of the KAR3 Gene

(A) Restriction map of the 4.6 kb KAR3 complementing region defined by overlapping genomic clones. The left end of the fragment as shown is defined by a Sau3A site. The complete DNA sequence of the 3.4 kb Pstl-BamHI fragment was determined. The boxed region indicates the open reading frame corresponding to the KAR3 gene. Restriction endonuclease sites are as follows: 8, Bglll; Ba. BamHI; H. Hpal; Hd. Hindlll; M, Mlul; N, Nrul; P, Pstl; R, EcoRI. (6) Predicted structural features of the putative KAR3 protein. Hydrophilicity (Kyte and Doolittle, 1962) and secondary structure (Garnier et al., 1976) predictions were made using UWGCG sequence analysis programs (Devereux et al., 1964). A graphic representation of the output of the program Peptide Structure as plotted by PlotStructure is shown (Wolf et al., 1966). In the upper plot, regions predicted to be hydrophilic are above the axis. For secondary structure, regions predicted to be in 5 turns, c helices. or 5 sheets are indicated in the appropriate graph by an elevated line. Those regions for which no specific structure is assigned are predicted to be random coil. Amino acid coordinates are shown below. (C) KAR3disruption alleles. Regions indicated by theopen boxes were deleted and replaced with the yeast LEU2 gene. Scale is the same as in (B).

Isolation of the KAR3 Gene The kar3-7 mutation (Polaina and Conde, 1982) causes both slow mitotic growth and a semidominant karyogamy defect. The wild-type KAR3 gene was isolated by complementation of the karyogamy defect. A kar3-7 strain (MY1124) was transformed with a centromere-based yeast genomic plasmid library (Rose et al., 1987) and approximately 18,000 transformants were screened by an interrupted mating assay (Rose and Fink, 1987). Five independent candidates were obtained that showed suppression of both the karyogamy defect (e.g., MY1258; Table 1) and the slow growth associated with kar3-7. The complementing plasmids defined a 4.8 kb overlapping segment of DNA (Figure 1A). To confirm the identity of the complementing region, the DNA fragment was used to direct plasmid integration by homologous recombination. Upon integration, plasmid pMR808 was found to be completely linked to the authentic KAR3 locus (100% parental ditypes in 32 tetrads). Therefore, the complementing DNA was derived from the authentic KAR3 locus. The KAR3 gene was assigned to the right arm of chromosome 18 by hybridization to electrophoretically separated yeast chromosomes and chromosome fragments. Subsequent genetic mapping (Table 2) placed KAR3 7 CM distal to Ty7-48. The gene order is CEN76 . AR07. . TEF7 . . RAD56. Tyl-48. KAR3. Thus, KAR3 defines a new gene that is the most distal marker on this arm of chromosome 18.

Kinesin-Related 1031

Table

2. Genetic

Cross KAR3 KAR3 KAR3 KAR3 RAD56

x x x x

AROP TEW RADSsd Tyl-48e x Tyl-48’

Karyogamy

Mapping

Gene

of the KAR3

Locus

PD

NPD

T

Linkagea

110 16 52 171 62

26 0 0 0 0

276 10 14 27 4

52 25 II 7 3

CM CM CM CM CM

aDetermined by the formula of Perkins (1949). Qmplled data from crosses MY1672 x K361-9D, MY2004 x MY2031, MY2004 x L3464, MY2067 x L3464, MY2107 x MY2026, MY2254 x MY2195, MY2161 x MY2217, and MY2004 x MY2131. =Data from cross between MY2254 and MY2195. dData from cross between MY2161 and MY2217. %ompiled data from crosses MY2161 x MY2217 and MY2004 x MY2131. Compiled data from crosses MY2161 x MY2217 and MY2162 x MY2216.

The Predicted KAR3 Protein is Related to Drosophila Kinesin Heavy Chain The smallest subclone that showed orientation-independent complementing activity was a 3.4 kb Pstl-BamHI fragment. The DNA sequence of this fragment revealed a 2187 bp open reading frame that potentially encodes a 729 amino acid protein of 84 kd (Figure 2A). The predicted KAR3 protein contains a region showing extensive homology to a domain of Drosophila kinesin heavy chain (Figure 26). Overall, the two regions are 38.5% identical (residues 388-716 of KAR3 versus 12-326 of kinesin). In kinesin this region corresponds to the globular mechanochemical domain responsible for ATPdependent microtubule movement in vitro (L. S. 8. Goldstein, personal communication). Amino acids 86106 of kinesin heavy chain include a sequence matching the ATP binding site consensus GX~GKTX#/ proposed by Walker et al. (1982). The KAR3 sequence between residue 468 and residue 488 contains a consensus ATP binding site and is 62% identical to kinesin. Furthermore, the carboxy-terminal portions of the homologous domains are 50% identical (residues 585-716 of KAR3 compared with 197-326 of kinesin). This region presumably includes the sites required for microtubule interaction in kinesin. Secondary structure analysis (Garnier et al., 1978) of the predicted KAR3 protein suggested that, like kinesin, KAR3 comprises three distinct domains. The sequence between residues 110 and 385 is hydrophilic and, with the exception of two short stretches between residues 235 and 295, is predicted to form an extended a-helical domain (Figure 1B). The presence of heptad repeats of hydrophobic residues (Figure 2A) suggests that this middle domain forms an extended a-helical coiled coil interrupted by a non-a-helical region. The non-a-helical region includes two proline residues. This pattern is reminiscent of kinesin, in which a flexible kink interrupts the central coiled-coil domain (Hirokawa et al., 1989; Yang et al., 1989). The smaller (l-109) amino-terminal globular domain of KAR3 is not homologous with kinesin or any other previously reported protein. However, this region is hydrophilic,

basic (21% Arg, Lys, and His versus 5.5% Asp and Glu; calculated pl of 12), and proline rich (8.2%), features shared by the microtubule binding domains of tau (Lee et al., 1989) and MAP2 (Lewis et al., 1988). This similarity may be significant since the localization of hybrid proteins (see below) suggests that the amino-terminal half of KAR3 contains a microtubule association domain. Based on the observed homology to kinesin heavy chain, we speculate that the carboxy-terminal domain of KAR3 interacts with microtubules and that KAR3 is a motility protein. However, KAR3 is unlikely to be the yeast kinesin homolog because of its different structural organization and the fact that the small globular domains are unrelated. KAR3 Is Induced by Mating Pheromone A DNA fragment internal to the coding region was used as a KAR3-specific probe for Northern analysis of total RNA. This probe recognized an mRNA of 2.7 kb that is present in both wild-type haploid and diploid (data not shown) strains during vegetative growth (Figure 3). The steady-state level of KAR3 mRNA was found to increase approximately 20-fold in a MATa strain treated with the mating pheromone a factor. Induction by a factor can be a pheromone response or a consequence of cell cycle arrest. To distinguish between these possibilities, we made use of a temperaturesensitive mutation in the CDC28 gene that causes cell cycle arrest at the same point as a factor arrest (Hereford and Hartwell, 1974). RNA was isolated from a cdc28 mutant strain (MY225) incubated at either 23% or 35°C. The level of KAR3 mRNA was not increased at 35% (Figure 3, lane 6). In contrast, subsequent treatment of the arrested cells with a factor did cause induction (lane 7). Thus, induction is pheromone specific and is not simply a consequence of Gl arrest. The basal-level transcription initiation site was mapped by RNAase protection and found to lie approximately 140 bp 5’ of the predicted initiation codon ATG of the KAR3 gene (data not shown). The induced transcripts were smaller than the basal-level transcript owing to initiation closer to the structural gene, approximately 70 and 40 bp upstream of the ATG (Figure 2A). The induction of KAR3 by a factor is consistent with the presence of several pheromone response elements in the KAR3 sequence. Pheromone response element sequences (ATTTGAAACA) are present in two or more copies and in either orientation in the 5’ noncoding regions of all known pheromone-inducible genes (Kronstad et al., 1987; Cross et al., 1988). Five close matches (7/s) occur in the KAff3 upstream region (-579, -294, -111, -61, and -42) along with several more degenerate (6/8) sequences (-580, -358, and -250). In addition, seven 718 matches occur within the coding region between +419 and +768. The importance of these elements to KAR3 induction has not been tested. KAR3 is the first pheromone-inducible gene that has been found to be expressed in both haploids and diploids. The expression of KAR3 in mitotic cells is consistent with the growth defect exhibited by kar3-7 and kar3 null mu-

Cell 1032

A CTGCAGCAGAAMTCCAGTAtAACCATCATCATGTTTGCTGTTTTTCGATTTTTTCTTTCTTGGGMGTCGTCGTCCTCTTCTTCTTCATCATCATCTTCTTCAGCATCACTTTG

-715 -600

TTCGTTATCTATMTTTTACATGATTCATCGCTAtACCTATTCTGCTCGTCTTCTTCGGCTTCATCACCTTCCATTATTGTATCTTTTTCCGGCTCATTACTTAACTCTTGGTTGCCACT

-480

ATTCCTTTTTTCACGCCCAAATTCTGCATTCTTTCTGGTTCTTTTCTTATCCTTAGTGTCTACTCTGTGCTTGGAGCCCATGATC~TTATGTACTGATTTTCCTTCGGCTTCTCTATCG

-360

CTTTATTCATAGCATCTGTTTATTACCTTTCCTTATATCTTATGGGCATCGAATCCTACATTTTTTTCTTICMMTTTTCCMTMGAGGGTMTGW\GATACACCAAAATGAATCTCA

-240

AACAAAATCAAMCMACACTGTTTACMTTTCATGCGCCTCGMTCAAMTATCATGATWGTATTACAGCTACAGCT~

-120

CGCTCTTTGTGTTTCTTATTATTCTATTTGMTATACCACAACMCTATC~~~GTCTTTGTTTMMMGGTAGATTTT~TMAGGACTTAGAGAMTTCTGGCAACTATTAAAGT 1 A:GG;AT

E

AC[rC

AC TA TC CA TG S8FSF

CA T TA GCAGCATCTCT GA AC AT GC EFEFa"LSFFER?NlTLE

GMT

TTATCCAATATTATATAAG~AATTAAACCTATCAATTTTTC

TA TTTAG TAAGAQTG CCflCvM E

121 C GC TA GCACA TCTTCTG CC GCAAC TA GG TA:TC$TA ACACT AT{AGjTA XaGA TC CAlAT CAAG’ AC TqrC 6StHF LLAjZbQafi? IHE 8 $1 E ES

AA AAdTA AA AAfTC$A iii fF

40

CC GCTTTTAAAGAATTATAAAGGTACA ~~LLKNYKGT

80

241 GE"6TT:GA:TTOTGEUi?CC~~~CA~CT~CG~TG:

C:~,,A,M,A,G~MIC,:A,~TG~G~~GG~~~AC~AG~T ,20 _____--__-______------~---G A GMMTC~TGECGTTAj3TCTC~TTTG~~CCTTC~T~A~~T~ATT~CAGCMCT~TTTGMMACAATGAA 361 ~[AC[CA(LA~TG~C[AA~G~~ ? 9 ..-...........--.--.--------------~--!--------~-----~-----~--------~--------------!--------~--?--?--~--~--~--~--~--~--~'60 G T TTTGT ~~GCgG~:CAJ~~G~AG r CA 'y 1AqU[ ~GCgGC~AGEGGF'S'~TA~G~GC~~~C~M~GG~AA~TG~ 2oo 481 C:~ATCTEY'~Y P F L 5 ___-.-___________-_------------------------.TTGAAATTTATGAAGA TAAACAGTTTGAMATGAAA AGCGT GCTTTTAGAT TAGAAG GGTM AAATAAAATCACCATGMCC TTCCACT 601 '~CA=AAFAA~AT'~C LKFNK~KGFENE8AELLDA#AAIE~“fiNKITNNSSr240 721 T:ACtGG

P

TGTTGAACGATGTTG ACAAAAGCATATGCTTGAAAAAGMGAATGGCUA A~GTOCCGATSGCAGTGGMGGATATAGAGCTGAATAATAAACATATGCAAGAA F P Y K K 0 IE L N N K H M Q E M L N 0 V c PKHNLEKEEUL

280

~4'ATcG:AAPCAT~GG~TCG~TA$AT:~CSTGBGT:GGliAG~G~Gc~c~~M~A~ ii9 !i ! ? !i T iz"v flAv fl

CC T CC CT T AC TA CA GTAA GTTA AGAAAAG E K 320 ____________-_______-----.------------.-------------.-----------.---------961 G:AGeGGf"F'AFMliGC:"~G~T~GG~GG~AT:MLS Cg~A~TA6'T:A~:"FTT~G~~T~A~C~T~G~T~TAT~~CA~TG~C~GG~TA~GA~G 360 ____.___________________________________.-----------------------------.-----------------------------------------------~~ 1081 GpGT:GnftTG~TTC:GATT~G~G~~ GGTTA v p C8CA $ATTGC L fi T~~~GT:AC~G GUM AG TA AC AGTTT TT TA GA TC TC AG TCTAAAAAATTTG 1 L 8 v L K N L 400 ________________________________________----------------------------1201 G AMTT

T 8 v? 8 8 T! f i S YGJYPT

TG TA TA CCTTA TAATGTT TG T T TGAC TA TG TGJTC$AT TAAG N Vq 1321 ATATTTGATCAACAGG TA AAATGTGG TGTTT TAAAGAAGTTGGTCAGTTAGTGCAAA rrgAr[AG#'G IFDPP~~NV~V~KEVGPLVP P

~NSIFBLT

p!c6

Dq

t t

~PIDEITSEFTE

1441

TC

1681 A GAfiTG$TA GA TT C?GC[rG F FPE 1801 AgTA:TT

FT

CA M:rCflrr[Gr

T

CT GG~G~~GGJGGr" fS TG

T Aq,rG

P

YGfiCA

E

TA:CT;CG

i

ATACGGACAAA AGGATCTGGGAAA Y E Q! G S $5 480

CMT TTAAAGA AAAAG AT GGATTATAAAGTTAACTGCGAATTCATT N"'LSLKFK88 0 Y K V N C E F 1520

AA CATTG CTTAAAGCACGAAATAC TCATGATCAGGAAA TAAGACTA CACGATA FIIELKHEI~HDoEFKTFII~~~

s

F

AA AA TG AGEAC;CT GTOTGECA AC:AAdTC:TGJT

T $jTTTM L 2TT E CT:AG 8Tc;CG;rA]rCflrG

2041 AFATPCC:ACTGC~TPTTtiACrCAFTG~G~TT~~~

r+ryTG;U

TG AT CApAT TGATAAA i T ! 0 K 440

AA CCT TTAA AT CA CC TA CA AGCAT AAATGAGCATT CTCCCGTTCACAC TTL~~E~~"SL~~FE~FA~NEHESRSH~OO

SFS EPFE

1921 GgrA~AT:AAF~$AC~~TAT~T~

2161 A(I'TE'AfCAF':GG:'A~TA~

TAATAdTqTA

&

4

T

G TGATG TATCA TC CT CA AA AT TCflTA:A’:‘qCTfiGA

1561 GAGATCTACMCGAGAACA CGTAGACTTATTGA EIYNENTVDLL~~?N

AC~~CA~AGEGC~AG$GC

ATZMAGT

F

TG$qCA:TI

"%

U:GGiCGgU

E

CGpA;AA;qTG&CT

E

TCAAGTTGTAGGG P V V G 640

TTTAG TCAGC TG TA TA CA AA ACiTA;AC GT;CA GAACTCAAAACTG i L 8 P 6 8 P? P 8 1 8 N S K L 680

SFOSEYiT AC AA CT CT T

TA rA+rG:GA

F

TC[CAftTT

4

GT[AAiAT;TG

EE

GT TAAAGTG K V 720

TGAGGTCMGGCCTTTTCTGGTCTTTTTCACTCCTTTGA~TGACA~GACTGTCCATACATTCATCACATGTAACTATATTATATATGAA a

729

2201 ACTCATTTTMTGCGCACACATAAMAGCMACTMCTMG~M~~~ATTTGTTATGTMMATGACCTCATACATGCTAGTATTTACACGAA~TTAATTGCTTAAATTTCAATCATCCTTA 2401 CCC~TTGGTTTACCCTCTGCAGGCAGAMCTTTTGCATCCTCCTTATTGCCCAATTTTCGCUATCACTTTMCATCTGGGTCCCATTTACCTTCCGTGGTGTTGAACCGCTTCCACCAT 2521 GAGGGGGATTTGMCCTAGGGTCTTCGCGTGG~MTTTGCCAA~C~~GCGCC~Cl~~ACCA 2641 TATTTCMTTCGTCIGACAGGTCGTTAGGA~TTTTGGGATCATAGTATTTTTCMCCAMTGTGTCCATTCTTTTCTATACCTGTCGATTAAATCATCATTTAAAGGATCC

B KAR3 Kinesin

380 390 400 410 420 430 440 450 460 470 480 . ..TLHNELDELRGNIRVYCRlRPALKNLENSDTSLIWVYVFKEVGD-LVGSSLDGYNVCIFAYGD~GSGK~F~M , ..:.: ::.:: :.. :....: . :. : .: . . . . . . . .. .. .. . . . . . . . . .:..:... .: . :.::: :::::::.:::: :: MSAERElPAEDSlKWCRFRP-LNDSEEKAGSKFWK-FPNNVEENCISI-----"" AGKV--YLFDKVFKPNASPE~YNEMKSIVTDVLAGYNG~lFAY~PTSS~HTM 10 20 30 40 50 60 70 80 90 100

490 500 510 520 530 540 550 560 570 580 590 600 -----LNPGDGIIPSTlSHlFNYINKLKTKGUDYKVNCEFIEIYNENIWLLR~NNNKED~SlGLKHElRHDDE~K~~~I~NVlSCKLESEEMVElILKKANKLRSTASTASNEHSSRSHSIFI . .. .. .. .. . . . . . .. .. .. . . . . . . . . .. .. .. ..: ::: . . .. . . :....... . . . .. . ..: : : . . . . . . . : .: :. .. .. .. .. .. .. .. .. .. . . . EGV~GDdVKoGIlPRIVNDlFNHIYA~~~~-LEFHIKVSYYEIYNDKIRDLL------DVSKVNL---SVHEDKNRVPYVKGATERFVSSPEDVFEVlEEGKSNRHIAVTNMNEHSSRSHSVFL 110 120 130 140 150 160 170 180 190 200 210 610 620 630 640 650 66D 670 680 690 700 710 729 720 IHLSGSNAKTGAHSYGTLNLVDLAGSERINVSPWGDRLRE~GNlNKSLSCL~VIHAL~PDS~KRHlPFRNSKL~YLLDYSL~WSK~LMFVNISPSSSHINE~LNSLRFASKVNS~RLVSRK~ :... .: . . . :.: ::::::::... . . :. : :..::::::: ::.:: :: .:..: :::.:.:::: .:: ::.:...: . . ::.: . .:: ..: :........ : lNVKPENLENPKKLSGKLYLVDLAGSEKVSKTGAEGTVLDEAKNINKSLSALGNVISAL--ADGNK~HlPYRDSKL~RILGESLGGNAR~~lVlCCSPASFNESE~KS~LDFGRRAK~VKNVVCVN... 220 230 240 250 260 270 280 300 310 320 290 330 340 Figure

2. DNA

and Predicted

Protein

Sequence

of KARI

and Comparison

with

Kinesin

Heavy

Chain

Sequence

(A) DNA and protein sequence. DNA sequence of the 3466 bp Pstl-BamHI fragment is shown; numbers start at the first ATG in the open reading frame and are indicated to the left. The corresponding protein sequence is numbered on the right. Upstream DNA sequences that match (irle) the consensus pheromone responseelement(Kronstad etal., 1987)are underlined. Approximate initiation sitesofthe basal (>)and pheromone-induced (>>)transcripts are indicated below the sequence. Amino acid residues in the putative ATP binding/hydrolysis site are indicated by asterisks. The predictedcentrala-helicaldomain isdelimited bycaratsataminoacids110and365.Regionswithinthea-helicaldomainthatcontain heptad repeats of hydrophobic residues are indicated by the dashed lines below the sequence. The termination codon is marked by the filled box. (B)Comparison of the predicted KAR3 carboxy-terminal domain with the mechanochemicaldomain of Drosophila kinesin heavy chain. Protein sequences were compared using the FASTP algorithm of Lipman and Pearson (1965). Identities (38.5% over 314 residues of kinesin) are indicated by two dots and similarities are indicated by single dots. Gaps introduced to optimize the alignment are indicated by dashes. Residues comprising the putative ATP binding site in kinesin are underlined. Drosophila kinesin heavy chain sequence is from Yang et al. (1989).

Kinesin-Related 1033

Karyogamy

Gene

+

odc28

30%

23W

35% Ohr

auk?

-

I

+ I

I

2

3

3hr

-II

+ I

I

+ I

4

5

6

7

KAR3=

TUB2 1 Figure

3. Induction

of KAR3 mRNA

by Q Factor

A Northern blot of total RNA was probed using a DNA fragment from the KAW gene and a probe specific to the 6-tubulin gene. The KAR3 probe recognizes an approximately 2.7 kb RNA in vegetative cells. Treatment with Q factor caused induction of a 2.6 kb transcript. Both transcripts are reduced in size in a /rar3-108::LEfJ2 strain (data not shown). The faint band above the KAR3 transcripts is due to nonspecific hybridization at the position of the 26s ribosomal RNA. Wildtype cells (MSlO) were grown at 30%. The c&28 mutant (MY225) was grown at 23% and a portion of the culture was shifted to 35% for the designated times. Cells were subsequently treated with a factor for 2 hr at the indicated temperatures.

tants (see below). However, KAR3 is the only gene known to be required for nuclear fusion that is induced by mating pheromone. This result suggests that KAR3 plays a direct role in the pheromone-dependent potentiation of nuclear fusion. KARI Is Essential for Karyogamy The original kar3-7 mutation is semidominant (Polaina and Conde, 1982). To determine the phenotype conferred by a kar3 null allele, several disruptions were constructed by replacing internal KAR3 fragments with the yeast LEUP gene (Figure 1C). A DNA fragment containing kar37OO::LEU2 was used to directly transform a wild-type haploid strain (MSlO) to Leu+. Similarly, fragments bearing either kar34Ol::LEUP or kar3-702::LElJP were used to transform a wild-type diploid strain (MS810). The diploid transformants were subsequently sporulated and dissected. Using either approach, it was possible to obtain viable Leu+ haploids in which the KAR3 locus was disrupted. The structures of the disrupted loci were confirmed by Southern blot hybridization (data not shown). Thus, the KAR3 gene is not essential for mitotic growth. However, like kar3-7, the disruptions of KAR3 resulted in slow mitotic growth. The effects in mitosis are discussed below. The nuclear fusion phenotype of kar3 disruption mutants differed markedly from the phenotype of kar3-7 mutants. When a kar3-7 mutant strain was mated to wild type, nuclear fusion was reduced about 30-fold (Table 1, line 2) as shown by reduced diploid formation and increased cytoductant formation. However, when kar3-7OO::LEU2 strains were mated to wild type (lines 4 and 5) nuclear fusion was comparable to that in a wild-type cross (line 1). In contrast, in a mating between two kar3-700::LfU2 strains (line 8) nuclear fusion was essentially abolished. Zygote formation occurred with normal frequency, as determined by phase-contrast microscopy. Loss of KAR3

Figure 4. Morphological Mutant Zygotes

Comparison

of Wild-Type

and /rar3-107::LEU2

Mating mixtures of wild-type strains MS10 and MS52 (A-C) or kar3701::LEU2 mutant strains MS524 and MS531 (D-F) were prepared and fixed for immunofluorescent staining. Early zygotes were stained with DAPI (Band E) to determine the positions of the nuclei and with an antibody to yeast f%tubulin (C and F) to determine the morphology of the microtubules. (A) and (D) are the corresponding phase-contrast images In the wild-type zygotes, nuclei have fused (upper left), are fusing (upper right), or are prior to fusion (bottom) with the SPBs closely apposed. In the mutant zygotes, the nuclei are separate and the long cy toplasmic microtubules fail to interconnect the two nuclei.

function resulted in the most severe karyogamy defect of any mutation reported to affect nuclear fusion. The crosses between disruption mutants and wild-type strains demonstrated that the wild-type gene product from a single parent is sufficient for normal frequencies of nuclear fusion. In contrast, in crosses to the kar3-7 mutant, the wild-type protein failed to promote efficient nuclear fusion. Therefore the kar3-7 protein poisons nuclear fusion, apparently by interfering with the function of the wild-type gene product. This is consistent with the semidominant character of the kar3-7 mutation. To characterize the severe karyogamy defect, wild-type and kar3-707::LEU2 zygotes were compared using fluorescence microscopy. In wild-type matings, karyogamy rapidly followed cell fusion. Early zygotes typically contained either a single fused nucleus or two closely associated nuclei with a high density of tubulin staining between them (Figures 4A-4C; see also Huffaker et al., 1988). In contrast, kar3-707::LEU2 zygotes (Figures 4D-4F) always contained two separated nuclei. Both nuclei were associated with long cytoplasmic microtubules that typically

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Figure 5. KA/?3-/acZ Hybrid Protein Localizes to and Stabilizes the Cytoplasmic Microtubules In (A)-(D), a wild-type strain (MSlO) containing plasmid pMR1390 (E. coli /acZ fused after the amino-terminal 309 codons of KAR3) was treated with o factor for 2 hr and prepared for immunofluorescent staining. In (E) and (F) nocodazole was added to 15 &ml at the time of a factor. In (G) and (H), nocodazole was added after a 2 hr preincubation in a factor. The cells were further incubated for 1 hr in nocodazole and a factor. In (I), the control wild-type strain MS10 containing the vector plasmid pMR79 alone was treated as for(G) and (H). (A) is a phase-contrast image; (B), (E), and (G) show staining with a monoclonal antibody against 5galactosidase; (C), (F), (H), and (I) show staining with rabbit anti+tubulin; (D) shows DAPI staining.

failed to interconnect. The lack of interaction suggests that KAR3 may mediate a lateral association between the antiparallel cytoplasmic microtubules. The kar3-7 Mutation Is in the Putative ATP Binding Site of the Kinesin-Related Domain Kinesin shows an ATP-sensitive association with microtubules in vitro (Vale et al., 1985). In the absence of ATP or in the presence of nonhydrolyzable ATP analogs (e.g., AMP-PNP), kinesin shows rigor binding to microtubules. ATP hydrolysis is required for release of kinesin from microtubules. Therefore, mutations in a kinesin-like motor protein that alter ATP binding or hydrolysis should create a protein that binds microtubules in a rigor fashion. The mutant protein is expected to physically impede the progress of the wild-type motor; such a mutation would be dominant. In light of this prediction, it was of interest to determine the nature of the semidominant kar3-7 mutation. kar3-7 was recovered from the genome (see Experimental Procedures) and mapped by in vivo (Kunes et al., 1987) and in vitro

recombination

methods

to a 242 bp EcoRI-Hpal

frag-

ment (+1304 to +1545). DNA sequence analysis revealed a single point mutation (G to A) at +1438. The mutation changed Gly-479 to Glu, altering one of the most conserved residues within the putative ATP bindinglhydrolysis site (GX4GKT to GX4EKT). The dominant nature of this mutation is consistent with the carboxy-terminal domain of KAR3 being functionally related to the mechanochemical domain of kinesin. KAR34acZ Hybrid Gene Products Localize to Cytoplasmic Microtubules in Shmoos The phenotypes of kar3 mutants and the predicted

protein

sequence suggest that KAR3 is likely to interact with microtubules. To determine the subcellular localization of the KAR3 protein, two gene fusions were constructed between KAR3 and the Escherichia coli /acZ gene. Hybrid proteins were expressed from the normal KAR3 promoter on centromere-based plasmids. One gene fusion (pMR1359) contained essentially the entire KAR3 coding sequence, whereas the other (pMR1300) contained only the amino-terminal 309 codons. The hybrid proteins were visualized by indirect immunofluorescence using antibody directed against 8-galactosidase. Both hybrid proteins showed the same pattern of localization. Results for the smaller fusion are shown in Figure 5. In cells treated with a factor, the hybrid proteins colocalized with the cytoplasmic microtubules that extend into the shmoo projection (Figures 5A-5D). Staining was brightest at the distal end of the fiber. In addition, a fainter dot of staining was frequently (35%-45%) detected at the periphery of the nucleus coincident with the SPB. To show that the localization of KAR34acZ is dependent on the cytoplasmic microtubules, strains were simultaneously treated with a factor and the microtubule-destabilizing drug nocodazole. Nocodazole causes rapid depolymerization of both cytoplasmic and nuclear microtubules in yeast (Jacobs et al., 1988). In all shmoos formed under these conditions, tubulin staining was absent or reduced to a faint dot at the SPB (Figure 5F). In the fusion-bearing strains, no KAR34acZ staining was observed in the shmoo projections. However, a dot was frequently detected at the nuclear periphery coincident with residual tubulin staining (Figure 5E). Thus, localization of KAR34acZ hybrid gene products is dependent on the formation of intact cytoplasmic microtubules. Remarkably, the KAR34acZ hybrid proteins stabilized

b,hin~in-Related

Karyogamy

Gene

preformed cytoplasmic microtubules. In this experiment, strains were first arrested with a factor and then treated with nocodazole. In fusion-bearing strains, the hybrid proteins were coincident with nocodazole-resistant cytoplasmic microtubules (Figures 5G and 5H). lntranuclear microtubules were not detected, providing an internal control for the efficacy of the drug. Under the same conditions, no microtubules were detected in wild-type strains (Figure 51). Thus, KARS-/acZ hybrid proteins, but not authentic KAR3 protein, had the effect of selectively stabilizing cytoplasmic microtubules. The stabilization activity of the hybrid proteins provides a striking demonstration of the interaction of these proteins with cytoplasmic microtubules in vivo. Both microtubule localization and stabilization were observed for hybrid proteins containing either all of KAR3 or only the amino-terminal 309 residues. Moreover, essentially the same results were obtained with strains containing either the wild-type KAR3 gene or a disruption mutation. Therefore, hybrid protein localization is not dependent on copolymerization with wild-type KAR3 protein. These data imply that the amino-terminal portion of KAR3 contains a microtubule association domain distinct from the kinesin-like domain. KAR3 Protein Localizes to the Cytoplasmic Microtubules To determine the localization of authentic KAR3 protein in cells, an antibody was generated against the carboxyterminal 208 amino acids of the protein (see Experimental Procedures). We were unable to detect specific localization in wild-type cells that had been treated with a factor. However, we reasoned that the interaction of the wild-type protein with microtubules might be too evanescent to accumulate to detectable levels. Since the kar3-7 mutant protein was predicted to bind more stably to microtubules, immunofluorescence staining of a mutant strain (MS1119) was examined. In contrast to wild type, the antibody uniformly stained cytoplasmic microtubules in kar3-7 cells arrested with a factor (Figure 6). Western blots demonstrated that the level of the mutant kar3-7 protein was not elevated relative to wild type (data not shown). No staining was observed in uninduced vegetative cells of either strain. These localization data confirmed that KAR3 associates with microtubules, as initially suggested from the

results obtained with hybrid proteins. These results further indicated that the association with the cytoplasmic microtubules reflects the intrinsic affinity of the KAFf3 protein. Finally, the apparently stable association of kar3-7 protein with microtubules substantiates the model that KAR3 acts in a kinesin-like fashion. kar3 Mutants Exhibit a Mitotic Detect Both kar3::LEUP and kar3-7 strains grew more slowly than wild-type strains. The doubling times were 160 min for kar3-707::LEU2 (MS524) and 195 min for kar3-7 (MS1119) compared with 100 min for an isogenic KAR3+ strain (MSlO). This increase was due at least in part to the production of nonviable cells (40% versus 30%) with a single nucleus and a short mitotic spindle (Figure 7). In wild-type cultures, fewer than 1% of cells had this morphology. This phenotype is characteristic of cell cycle arrest in mitosis. Given the possible role of KAR3 protein in microtubule motility, these results are consistent with a defect in spindle elongation. Although KAff3 is not essential for viability, it plays an important role during mitosis. Discussion The KAR3 gene was originally identified as one of several genes required for nuclear fusion in S. cerevisiae (Polaina and Conde, 1982). DNA sequence analysis revealed that the predicted KAR3 protein comprises three structural domains: a carboxy-terminal domain homologous to the mechanochemical motor domain of Drosophila kinesin heavy chain, a central region predicted to form an extended a-helical coiled coil, and a small amino-terminal globular domain that is unrelated to kinesin. Protein localization data and the phenotypes of mutants suggest a predominant role for KAR3 protein in the function of the cytoplasmic microtubules. The structure of KAR3 implies the existence of a family of kinesin-like proteins characterized by a kinesin motor domain tethered to different protein binding domains. The various members of the family would translocate different macromolecules and organelles along microtubules. A third member of the family has recently been identified in

Cell 1036

Figure

7. &ar3-lOl::LEU2

Cells Are Partially

Defective

in Mitosis

A vegetative culture of a ker3-lOI::LEU2 strain (MS524) was fixed and stained with DAPI (B) and anti-f3-tubulin antibody(C). (A) is the corresponding phase-contrast image. The upper part of the figure shows two normal-appearing cells in late nuclear division. The lower part of the figure shows four cells from the same culture that are blocked in mitosis (large bud, undivided nucleus). The large arrows indicate cells blocked in spindle elongation. The small arrow indicates a cell in which mitosis appears blocked prior to spindle elongation. The abnormal cells make up 30% of the ceils present in the vegetative culture. Such cells are rarely seen (