saccharomyces cerevisiae pac2 functions with cznl, 2 and 4 ... · saccharomyces cerevisiae pac2...

Copyright 0 1997 by the Genetics Society of America

Saccharomyces cerevisiae PAC2 Functions With CZNl, 2 and 4 in a Pathway Leading to Normal Microtubule Stability

M. Andrew Hoyt, Jennifer P. Macke, B. Tibor Roberts and John R. Geiser

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 2121 8

Manuscript received January 7, 1997 Accepted for publication April 10, 1997

ABSTRACT The products of the Saccharomyces cerevisiae CINl, CIN2 and CIN4 genes participate in a nonessential

pathway required for normal microtubule function. In this article, we demonstrate that the product of PAC2 also functions in this pathway. PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cinlA, cin2A and cin4A. These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl. Involvement in a common functional pathway is indicated by the observation that all double mutant combinations are viable and no more affected than any single mutant. In addition, extra copies of CINl were found to suppress the benomyl sensitivity of pac2A, cin2A and cin4A, but not that caused by other mutations that affect microtubule function. Cinlp and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of P-tubulin into an assemblycompetent state. Alleles of CINI were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion. Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cinlp and regulates i ts tubulin folding activity.

M ICROTUBULES are ubiquitous fibrous elements in eukaryotic cells that perform essential roles

in cell architecture, division and intracellular transport. The major constituent of microtubules is the a@-tubulin heterodimer. A notable property of microtubule- based structures is their morphological heterogeneity and the rapid manner in which they disassemble and reassemble into new forms. These properties must be specified by the activities of the tubulin subunits and the numerous factors with which they interact. While some of the dynamic properties of microtubules can be reproduced in vitro using purified tubulin, it is likely that the morphology of most cellular microtubule structures is determined by the actions of the nontubulin factors.

The yeast Saccharomyces cerevisiae is a particularly trac- table organism for the study of microtubule function. In this organism microtubules are required solely for chromosome segregation (mitotic and meiotic spindle function) and for nuclear fusion during mating (karyo- gamy). There is a single and essential @-tubulin gene, TUB2, and two a-tubulin genes, TUBl and TUB3 (NEFF et al. 1983; SCHATZ et al. 1986a,b). The a-tubulin genes encode functionally interchangeable products but only TUBl is essential, presumably because it provides most of the a-tubulin for the cell. Mutant alleles of these genes cause observable defects in microtubule structures and often cause increased sensitivity to com-

Corresponding author: M. Andrew Hoyt, Department of Biology, Mudd Hall, The Johns Hopkins University, 3400 N. Charles St., Balti- more, MD 21218. E-mail: [email protected]

Genetics 146 849-857 (July, 1997)

pounds, such as benomyl, that promote microtubule disassembly (SCHATZ et al. 1986b; HUFFAKER et al. 1988; STEARNS and BOTSTEIN 1988).

Previous studies have identified three additional genes that do not encode tubulin subunits but nonethe- less are associated with phenotypes very similar to those caused by tubulin mutants (HOW et al. 1990; STEARNS et al. 1990). CINl, CIN2 and CZN4 deletion mutants display extreme sensitivity to benomyl and are cold sensitive for viability. At the nonpermissive temperature most of the microtubules in these mutant cells disassembled. A number of genetic interactions between mutant alleles of these three genes and various tubulin alleles were observed (STEARNS et al. 1990). For example, the viable, but benomyl-sensitive tubl-1 allele could not be combined with any cinl , cin2 or cin4 mutant allele to yield viable cells. In contrast, all possible CINI, CIN2 and CIN4 double and triple mutant combinations were viable and were no more phenotypically affected than any single mutant. The similarity in phenotype between the single and multiple mutants suggested that the products of these three genes function together in a nonessential pathway leading to normal microtubule stability.

CIN4 deletion alleles ( c in4A) cause a slightly less severe phenotype than deletion of CINl or CIN2 (STEARNS et al. 1990; see Figure 1). Interestingly, the less-sensitive to benomyl phenotype of cin4A was found to be epistatic to the more sensitive phenotype of cin2A; c in2A cin4A double mutants displayed the less-sensitive cin4 phenotype. In contrast, c i n l A c i n 4 A double mutants displayed the more-sensitive cinl phenotype. The epis-

850 Hoyt et al.

tasis relationship so defined (cinl > cin4 > cin2) suggested the possibility that Cinlp acts closer to its target in this microtubule-stabilizing pathway. Among isolated cin4 point mutants, a subset exhibited a greater sensitivity to benomyl than that displayed by cin4A. CIN4 encodes a guanine nucleotide-binding protein most similar in sequence to members of the ADP-ribosylation factor (arQ family (BOTSTEIN et al. 1988). The highly benomyl-sensitive cin4 alleles encode forms with changes in conserved amino acids involved in guanine nucleotide-binding (T. STEARNS, C. CLARK, M. A. HOW, R. KAHN and D. BOTSTEIN, unpublished data). Taken together, it seems reasonable to propose that the microtubule-stabilizing activity of Cinlp is regulated by the upstream-acting Cin4p and Cin2p. The genetic observations reported herein provide strong support for this hypothesis.

In a recent article we described the identification of 20 S. cermisiae genes whose products become required in the absence of the nonessential kinesin-related mitotic motor Cin8p (GEISER et al. 1997). Cin8p participates in mitotic spindle assembly and is the most important spindle pole-separating motor in this organism. Among the mutants that perish in the absence of GIN8 (pac) , we found two that caused extreme sensitivity to benomyl. One was found to carry a mutant allele of CINl. The second defined a novel gene that we named PAC2. In this article we describe our characterization of PAC2 mutants and provide evidence that the product of this gene functions in the CINl, 2, 4 microtubule- stabilizing pathway. The products of PAC2 and CINl were found to be related by sequence to mammalian proteins that participate in the folding of P-tubulin into a microtubule assembly-competent form.

MATERIALS AND METHODS

Yeast strains and media: The S. cerevisiae strains used in these experiments are derivatives of S288C and are listed in Table 1. The isolation of the pac2-l mutant was described in GEISER et al. (1997). The cinl, 2 and 4 deletion alleles and cin4 missense alleles were described previously (STEARNS et al. 1990). The chromosomal CIN4 locus was marked with URA3 (CIN4:URA3) by inserting a genomic DNA fragment from directly adjacent to CIN4 into a URAkontaining integrating vector, cutting within this region and integrating by transformation. Rich (YPD), minimal (SD) and sporulation media were as described (SHERMAN et al. 1983). Benomyl (Dupont) was added to rich media from a 10 mg/ml stock in dimethyl sulphoxide.

Cloning and characterization of PAC2 and CINI: Strain MAY2758 (Table 1) is unable to remain viable without plasmid pMA1208 since it provides the CIN8 required due to the pac2-1 allele. This strain is rendered sensitive to cycloheximide due to the dominant CYH2 allele also present on the plasmid. A plasmid that confers upon MAY2758 the ability to segregate cycloheximide-resistant colonies was isolated from a S. cerevisiae genomic library constructed in a TRPl-CEN vector (gift from C . CONNELLY and P. HIETER). Subcloning and sequencing identified a previously uncharacterized gene capable of

TABLE 1

Yeast strains and plasmids

Genotype

Strains MAY589 MATa ura3 his3 leu2 ade2 MAY591 MATa ura3 his3 leu2 lys2 MAY755 MATa ura3 his3 leu2 t?pl tub3::TRPl MAY776 MATa ura3 his3 leu2 ade2 tubl-1 MAY1013 MATa ura3 his3 leu2 lys2 cin4-4 MAY1014 MATa ura3 his3 leu2 lys2 cin4::URA3 MAY1016 MATa ura3 his3 leu2 ade2 cin4-4 MAYlOl9 MATa ura3 his3 leu2 ade2 cinl::URA3 MAY1067 MATa ura3 his3 leu2 ade2 tub2-104 MAY1096 MATa ura3 his3 leu2 ade2 CIN4::URA3 MAY1 100 MATa ura3 his3 leu2 lys2 CIN4:URA3 MAY1237 MATa ura3 his3 leu2 lys2 cin4-4 cin1::HISj MAY1286 MATa ura3 his3 leu2 ade2 cinl::HIS3 MAY1298 MATa ura3 his3 leu2 ade2 cin2::LEUZ MAY2058 MATa ura3 his3 leu2 lys2 cin8::LEUZ MAY2758 MATa ura3 his3 leu2 ade2 trpl cyh2 pac2-1

MAY2853 MATa ura3 his3 leu2 lys2 pacP::URA3 MAY2854 MATa ura3 his3 leu2 ade2 pac2::URA3 MAY2863 MATa ura3 his3 leu2 lys2 cyh2 pac2-l

MAY2901 MATa ura3 his3 leu2 ade2 lys2 cyh2 pac2::URA3 MAY4384 MATa ura3 his3 leu2 t?pl pac2::URA3

pMA1071 GIN1 LEU2 CEN pMA1208 CIN8 CYH2 LEU2 CEN pMAl286 PAC2 TRPl CEN pTRlO GIN1 URA3 2p pTRl3 CINl LEU2 2p

cin8::HIS3 (pMA 1208)

cin8::HIS3 (pMA1208)

Plasmids

rescuing pad-1 contained on this plasmid (GenBank accession no. U16814). A deletion of PAC2was created by removing the DNA between XhoI (base pair 469) and HpaI (base pair 1'136) within the open reading frame and replacing it with DNA encoding URA3. This deletion caused a high sensitivity to benomyl (no growth in media containing 5 pg/ml). MAY2901, a pac2::URA3strain, was crossed to MAY2863 (pac2- 1 ) . All spores in 16 tetrads were benomyl sensitive, indicating that all tetrads were parental ditypes and pac2::URA3 is linked to pac2-1.

With the recognition that pac2 alleles cause sensitivity to benomyl, the pac mutant collection was screened for other mutants with this phenotype. Another benomyl-sensitive mutant was recovered and mated with characterized benomyl- sensitive mutants (STEARNS et al. 1990). Noncomplementation with a cinl::HIS3 mutant was observed. This diploid was sporulated. All viable progeny from the 14 tetrads examined were found to exhibit the benomyl-sensitive phenotype, indicating linkage of the pac mutation to CINl. The cloning of CINl was described previously (STEARNS et al. 1990). The determined DNA sequence of CINl is reported in GenBank accession no. 246420.

Microscopy analysis: Log phase cells incubated at 26" were shifted to 11" for 24 hr, fixed, prepared for immunofluores- cence microscopy and stained for tubulin using the mono- clonal YOL1/34 antibody (Bioproducts for Science) and for DNA with 4,6-diamidino-2-phenylindole (DAPI; Sigma) as previously described (Horn et al. 1990).

Microtubule-Stabilizing Pathway 8.5 1

Benomyl (pg/ml) benomyl-sensitive puc mutants were killed by as little as 2.5 pg/ml. Many mutants have been described that

0 1 2.5 5 10 15 display benomyl sensitivity but only defects in six genes

wild-type

pac2-1

pac2-1 pCINl-2p

pac2A

pac2A pCINl-2p

cinlA

cin2A

cin2A pCIN1-2p

cin4A

cin4A pCINl-2p

tub1 -1

tubl-1 PCINI-2~

tub3A

t ~ b 3 A pCINl-2p

FIGURE 1.-Benomyl scnsitivity of various genotypes. H a p loid cells of the indicated genotypes were suspended in water and spotted on rich media containing the indicated benomyl concentration. pCIN1-2p. CINl-2p plasmid transformant.

Suppression of cin4-4 Independent colonies of cin4-4 strains MAYlOlJ and MAY1016 were picked from rich agar plates, resuspended in water and individually plated on rich media containing 5 pg/mI benomyl. At this concentration, resistant colonies arose at a frequency of approximately one in 10’ cells plated. To ensure that the suppressor mutations occurred independently, only a single revertant was picked from each original colony.

CINI alleles that could suppress cin4-4 were recovered from the chromosome by the g;lp repair method (ROTHSTEIN 1991). Plasmid pMA1071 was gapped with Smnl and BnmHI removing most of the CINI open reading frame. Recovered plasmids with the gap healed by recombination were trans- formed into MAY1237 (c in lA r in4-4) . Transformants were able to grow on 5 p,g/ml benomyl, indicating that the sup pressing alleles had been recovered. The entire SndX.ITX12011206 internal fragment of CINI was sequenced for each suppressor allele and a single change was found within each.

RESULTS

pac2 mutant phenotypes and interactions resemble n’nl, 2 and 4: Among the pnc mutants unable to survive loss of the nonessential CIN8 gene (GEISER rt nl. 1997), we found two that caused extreme sensitivity to the antimicrotubule compound benomyl. Our wild-type haploid strain is resistant to an approximate maximum of 15 pg/ml benomyl in the media (Figure 1). The two

were known to cause sensitivity to ~5 pg/ml, TUBI, 2 and 3 and CINI, 2and 4 (STEARNS el nl. 1990). Comple- mentation and linkage analysis (see MATERIAIS AND METHODS) revealed that one of the benomyl-sensitive pac mutants carried a defective allele of CINI. Cloning and linkage studies (see MATERIAIS AND METHODS and below) revealed that the second mutant, im2-I, carried a defect in a previously uncharacterized gene. We created a deletion allele of PAC2 by substituting part of the gene with UM3. The pac2A haploid strains created were viable, indicating that pac2 is a nonessential gene. A viable pnc2A cin8A strain was created by covering the cin8 defect with a CINkontaining plasmid. This strain was unable to survive plasmid loss, indicating that the pnc2A allele caused the same perish-in-the-absence of ClN8 phenotype caused by pnc2-I. The microtubule defect (see below) of the deletion strain was more severe than the pnc2-1 mutant. All further characterization experiments were performed with the deletion allele.

As described previously (STEARNS pt nl. 1990) cinlA and cin2A mutants were found to be slightly more sensitive to benomyl than the cin.4A mutant (Figure 1). The sensitivity of the pnc2A mutant was found to lie between the very sensitive cinlA and cin2A and the less-sensitive cin4A. These slight differences were highly reproducible but their cause is unknown. Interestingly, the less sensitive phenotype of cin,4A was previously found to be epistatic to the more-sensitive phenotype of cin2A (STEARNS et nl. 1990). All double mutant combinations of cinlA, 2A and 4A with pac2A were created and found to be viable. Unlike the cin2A cin4A situation, the benomyl sensitivities of the created pnc2A double mutants were the same as the most sensitive single mutant in each combination (data not shown).

It has been observed that the combination of cinl, 2 or 4 alleles with the cold- and benomyl-sensitive tuhl-1 allele causes inviability (STEARNS et nl. 1990). Similarly, we have found that the pac2A tubl-1 double mutant is inviable. Across between a pac2::URA3strain and a tuhl- 1 strain yielded eight tetrads with three viable spores (tetratypes) and one with two (a nonparental ditype). The genotype of the dead spore in these tetrads could be deduced from the phenotypes of the viable spores. In each case the dead spore was determined to be the pnc2::UR43 tubl-1 double mutant, indicating this combination is inviable or unable to survive spore germina- tion. In another series of crosses, we observed that spores deleted for the nonessential a-tubulin gene TUB3and either PAC2or CINI were unable to grow into colonies. Finally, like pnc2A and cinlA, cin2A could not be combined with cin8A to yield viable spores. Unex- pectedly, we could recover viable cin4A cin8A double mutant spores that formed colonies approximately half

852 Hoyt et al.

TABLE 2

Viability of double mutant combinations

pac2A cinlA cin2A cin4A tubl-1 tub3A cin8A

pac2A - V" V V I I I cinlA - V V I I I cin2A - V I NT' I cin4A - I NT V

Assessed by the ability of double mutant spores to germinate and grow into colonies. " Viable.

Inviable. Not tested.

the size of wild-type or single mutants. This may reflect the less severe nature of the cin4A phenotype. The viability of the various mutant combinations examined is summarized in Table 2.

At low incubation temperatures cinl , 2 and 4 mutants disassemble most cellular microtubule structures and arrest growth as large-budded mononucleate cells (HOW et al. 1990). pac2A and c i n l A mutants were com- pared morphologically with the wild type following growth at either 26 or 11". At 26", the mutants grew well and displayed a distribution of morphologies that was not greatly different from the wild type (Figure 2). The two mutants displayed a slight elevation in large- budded mononucleate cells and a corresponding slight reduction in large-budded cells that had completed nuclear DNA segregation. After incubation at ll" for 24 hr, however, these differences became much more prominent. Seventy-four percent and 75% of the pac2A and cinlA cells, respectively, were large-budded and most were mononucleate. Antitubulin immunofluo- rescence microscopy revealed that, similar to c i n l A cells, the microtubules in the pac2A cells had mostly disassembled at 11" (Figure 3). In many pac2A and c i n l A cells, a small amount of residual microtubule structure was found associated with the periphery of the nucleus. This presumably represents somewhat more stable microtubules associated with the microtubule or- ganizing center (the "Spindle Pole Body"). The microtubule structures in mutant cells grown at 26" appeared indistinguishable from those in wild-type cells (not shown).

The microtubule defects observed for the pac2 strains could reflect a reduction in the abundance of one or more of the tubulin polypeptides. However, Western blots of total cell protein extracts, probed with antibod- ies specific for Tublp, Tub2p or Tub3p (HOW et al. 1990), revealed no differences in levels of these proteins between pac2A and wild type (data not shown).

PAC2 and CZNl encode homologues of mammalian factors required for p-tubulin folding: PAC2 was cloned by complementation of the perish-in-the-absence of CZN8 phenotype (see MATERIALS AND METH- ODS). The clone obtained was subsequently demon-

strated to complement the benomyl-sensitive phenotype of pac2. Subcloning was performed to identify the complementing open reading frame. A chromosomal deletion allele of this gene was created and crossed to a pac2-1 strain. When this diploid was sporulated, all spores examined from 16 tetrads were found to be benomyl sensitive, indicating linkage of the cloned DNA to the PAC2 locus. The cloning of CINl was described previously ( STEARNS et al. 1990).

CINl and PAC2 encode polypeptides of predicted molecular weights 117 and 59 kD, respectively. A data- base homology search yielded three gene products with sequence similarity to Cinlp (between 20 and 24% identical and 45 and 47% similar): bovine cofactor D (133 kD; GenBank accession no. U61233), which participates in the in vitro folding of P-tubulin into an assembly-competent form (TIAN et al. 1996), and gene-products from C. elegans (140 kD; GenBank accession no. 278062) and S. pombe (126 kD; GenBank accession no. Z69379), both of unknown function. Pac2p was found to be similar (30% identical and 54% similar) to human cofactor E (59 kD; GenBank accession no. U61232), which also participates in the P-tubulin folding process (TIAN et al. 1996). A homologue of Pac2p has also been found in S. pombe (E. GRISHCHUK and J. R. MCINTOSH, personal communication). The amino termini of Pac2p and cofactor E (Figure 4) contain a close match to a sequence that forms a microtubule-binding domain in a number of other microtubule-associated proteins, in- cluding CLIP170, the dynactin p150G'""d subunit (Pac13p/Nip100p in S. cumisiae) and S. cumisiae Biklp/Pacl4p (RIEHEMANN and SORG 1993; GEISER el al. 1997).

Extra CZNl suppresses cin2, cin4 and pac2 We found that extra copies of CZNl could specifically suppress the benomyl sensitivity of cin2, cin4 and pac2 mutants. High- copy plasmids (2p based) carrying CINl were trans- formed into c i n 2 4 c i n 4 A , pac2-1 and pac2A. The re- sulting strains were found to be significantly more resistant to benomyl than the untransformed mutants (Fig- ure 1 ) . Resistance to benomyl was not restored to wild- type levels, however. The effect of extra CZNl was specific to these three mutants and did not represent a

A

wild-type

pac2A B Unbudded Small-budded Large-budded

43 29 28 27 33 41

cinld 33 30 37

X.53

DNA tubulin

50 23 28 6 74

cinld 16 10 75

1 17 82 0 6 39 53 2

cinlA 2 38 58 2

wt 3 7 8 8 2 11' pac2A 66 24 7 3

ChlA 63 29 6 2

general increase in benomyl resistmcr. Transformation of the 2p C I N I plasmid into the l,enom!~l-sensitive / 1 r b 1 - I and tuh?A strains had no effect (Figure 1 ) . Transfor- mation into the w i l d type also had no effect (not shown) and we note our prcviotls finding that grcatl!, overpro- duced Cinlp levels, from the high level galactose pro- moter, a c t d l y decreased the resistance o f thc, wild type to benomyl (HOIT p/ crl. 1990). Two-micron plasmids expressing CLV2 or CLY4 did not have an!. cffect upon benomyl resistance when trmsformed into thc YI ' 1 1 0 ~ 1 s .' mutants (not shown).

CINZ alleles that specifically suppress c in44 X s c t o f CIN4 mutant allelcs (rir74-4, -5, -i and -8) arc: rlnique i n that they canse a greater sensitivity t o lwnom!.l t h a n a deletion of CLV4 (Sre.\~ss p/ nl. 1990). This intlicatrs that the products o f these ;~lleles arc active within the cell and interact with other gcnr prodr~cts i n a tlrlctrri- o w manner. To explore the nature of this interaction. we collected mutmts that are suppressed for the ri174-4 benomyl-sensitive phcnotypr. Suppressor mrltants were selected on media containing 3 pg/ml Imlomyl and arose at a frequrncy o f approsimatcly one i n 10' cells

854 Hoyt et al.

Pac2p 5 I G D R L K I G G Y F C T I K F I G V I K P W P S ~ Y G ~ ~ D H S R G ~ S G T I D D I ~ F D V Q I P N S G S F 65

cofactor E 10 1GRRVEVNGEHATVRFAGWPPVAGPW.LGVEWDNPERGKHDGSHEGTFKCRHPTGGSF 69 I G R + + + G T++F GV+ P ++ GVEWD+ RGKH G+ + YF + P GSF

{ G G W. GV L .GKH GS G YF

CAP-Gly consensus Y I 1 N T F L V A

M M

F~CURE 4.-Comparison of the amino termini of S. cmvisiar! Pac2p and human cofactor E. Both match closely the CAP-Gly consensus sequence found in microtubule associated proteins (from RIEHEMANN and SORG 1993). In the line between Pac2p and cofactor E, identical amino acids are indicated by letter; conservative substitutions are indicated by a plus (+). A period (.) indicates a single amino acid gap introduced to maintain alignment.

plated. Eight suppressor mutants that arose indepen- high levels of benomyl (up to 50 pg/ml). Growth at this dently and exhibited Mendelian inheritance (2:2 segre- high level of inhibitor is usually indicative of a resistance gation) were chosen for further analysis. These s u p mutation in the 0-tubulin gene TUB2 (THOMAS et al. pressed strains all grew well on 5 pg/ml benomyl but 1985). Indeed, a cross to the benomyl-resistant tub2-104 did not grow as well as wild type on 10 pg/ml (Fig- allele revealed that this suppressor mutation was linked ure 5A). (all 15 tetrads analyzed were parental ditypes). In an-

As an early step in their analysis, these mutants were other cross we found that this suppressor mutation crossed to a CZW strain that was marked with URA3 at could raise the benomyl resistance of cin4A as well as the CZN4 locus (see MATERIALS AND METHODS). Among cin4-4. In this case, therefore, suppression was most the progeny of this cross we were able to identify CZN4 likely achieved nonspecifically by the creation of a cells that carry the suppressor mutation alone. For one tubulin dimer that is more resistant to the actions of mutant, all the progeny recovered from 10 tetrads were benomyl. able to grow well on 5 pg/ml benomyl, indicating that As described below, the remaining six suppressors the suppressor mutation was linked to cin4-4. There- were determined to be alleles of CZNl and are referred fore, this suppressor most likely arose by an intragenic to as CZNI-440 in Figures 5 and 6. All six mutants were mutation event. For another mutant, half of the Ura+ partially dominant for the suppressing phenotype con- progeny were able to grow on media containing very ferred (Figure 5B). In a cin4-4 homozygous diploid

A Benomyl (pglml) B Benomyl (pglml)

0 1 2.5 5 10 15 0 1 2.5 5 10

wild-type

cin4-4

cin4-4 CINl-4401

cin4-4 CINI -4402

cin4-4 CINl-4404

cin4-4 ClNl-4405 i cin4-4/cin4-4 CINI/CINI

cin4-4/cin4-4 CINI -4401KINI

cin4-4/cin4-4 CINI-4401/CINI -4401

cin4-4kin4-4 CINl-4402/CINl

cin4-4/cin4-4 CINI -4402/CIN1-4402

cin4-4/cin4-4 ClNl-44O4/CINl CINI -4401

CINI-4402 cin4-4/cin4-4 CINl-44O4/ClNI-4404

cin4A cin4-4/cin4-4 CINI -4405/CINl

cin4A CINl-4405 cin4-4/cin4-4 CINI -44OYCINI -4405

cin4A CINI -4402

cin4A CINl-4404

FIGURE 5.-CINI alleles specifically suppress the benomyl sensitivity of cin4-4. (A) Haploid cells of the indicated genotypes were spotted on rich media containing the indicated benomyl concentration. The slight reduction in growth of the cin4A C I M - 4405 spot at 2.5 pg/ml benomyl relative to other cin4 spots was neither reproducible nor significant. (B) The CINI alleles are dominant for suppressing ability. Diploid cells of the indicated genotypes were spotted on rich media containing the indicated benomyl concentration.

Microtubule-Stabilizing Pathway 855

CINl-4404 CINl-4406

0 I

I ... 1021 TGT CAT TTT CAG AAA TCT CAC TTT CAA ATC ATC AAG GGC CAC CAT

3 4 1 cys his phe gln lys ser his phe gln ile ile lys gly his his

=g

ATC AGG GAC TCG ACA AAC TTC ATT ATC TGG TCA GTA ATA AGG TCA ile arg asp ser thr asn phe ile ile trp ser val ile arg ser

AAC CGT AGC AAT AGT TTA TCG CCG CAG GTT CTA CAA TCA CTA CTT asn arg ser asn ser leu ser pro g ln Val leu gln ser leu leu

CINl-4405 CINl-44 02

CINl-4401 CINl-44 03 C T

TCT CAT CTG TTA ATA AAC GCC TTT TTT QAC CCC GAA TTA ATA ATC 1200.. ser his leu leu ile asn ala phe phe asp pro glu leu ile ile 400

I I C I

I I I

FIGURE 6.-The suppressing changes in CZNl cluster within a small region. The suppressing CZNl alleles were recovered from the genome by plasmid gap repair. The changes indicated were determined to be solely responsible for the suppressing phenotype.

Pro tyr

background, CIN1-44O/CINI always yielded higher resistance to benomyl than CINl/CINl, but lower than CINI- 440/CIN1-440. Intercrossing revealed that these six suppressing mutations were linked to one another. When crossed away from cin4-4, two of these mutations, CINI- 4401 and -4402, caused slight benomyl sensitivity on their own (Figure 5A). One of these mutants (cinl- 4402) was crossed to a cinl::HIS3 strain. All progeny from 12 tetrads displayed benomyl sensitivity (either the highly sensitive cinl::HIS3 phenotype or the slightly sensitive suppressor allele phenotype), indicating linkage. Therefore, this suppressor, and the five to which it is linked, represent mutant alleles of CINI.

A gapped CINl-CENplasmid was used to recover the mutant alleles from the chromosome by recombination (see MATERIALS AND METHODS). Sequencing of the mutant DNA recovered revealed a single nucleotide change for each suppressor allele within the 359-basep- air Sac1847-XhuIL206 internal fragment of CZNl (Figure 6). We demonstrated that these changes conferred suppressing activity by introducing the six mutant Sad-XhoI fragments into an otherwise wild-type CINI plasmid. Introduction of these plasmids into a cin4-4 cinlA strain yielded suppression. Although the suppressor mutants were selected independently, two pairs of mutants were found to have suffered the same nucleotide change (Figure 6) . For both of these, the mutation is the only possible single nucleotide change that yields the re- sulting amino acid change. The independent recovery of the same changes twice suggests that we are ap- proaching saturation with this screen and that only very specific changes in CINl will suppress. We also note with interest that the four suppressing changes within Cinlp are clustered within a 57 amino acid region (out of a total Cinlp size of 1014 amino acids).

The specificity for suppression activity was tested by crossing the four CZNI-440 mutants to the three other

th

cin4 mutants that, like cin4-4, cause high benomyl sensitivity and involve amino acids important for guanine nucleotide binding (cin4-5, -7 and -8). We also tested the ability of the four to suppress the less-sensitive phenotype of cin4A (Figure 5A). In addition, CINI-4401 and -4402 were crossed to cin2A, pac2A and pac2-1 strains. In none of these cases did we see any evidence for suppression of benomyl sensitivity among the double mutant cross progeny. Therefore, the CINI-440 alleles suppress cin4-4 in a highly specific manner.

DISCUSSION

We have described our characterization of the S. cere- visias PAC2 gene and demonstrated by genetic criteria that it functions along with CINI, 2 and 4 in a pathway leading to normal microtubule stability. pac2-1 and cinl- 20were identified as mutants that will not survive loss of the nonessential CIN8 mitotic motor gene. Presumably, this represents a reduced proficiency for the remaining mitotic motors to operate on unstable microtubules. pac2A cells displayed phenotypes extremely similar to cinlA, cin2A and cin4A cells. The growth of all these mutants was severely affected by low levels of benomyl in the media (<5 pg/ml). At low incubation temperatures all four mutants suffered massive loss of microtubule structure. This defect in microtubule structure caused a block to cell cycle progression typical of microtubule-disrupting treatments (HUFFAKER et al. 1988; JA- COBS et al. 1988). Similar to the CINI, 2 and 4 mutants, pac2A could not be viably combined with two a-tubulin mutations, tubl-1 and t u b 3 A , which affect microtubule function. However, all double mutant combinations in- volving cinl, 2 and 4 and pac2 are viable and no more sensitive to benomyl than any single mutant. This is the anticipated finding for the products of genes that act within a single, nonessential pathway.

856 Hoyt et al.

Of high significance is the finding that mammalian homologues of Cinlp and Pac2p contribute to the folding in vitro of @-tubulin into a form competent for assembly into tubulin dimers and microtubules (TIAN et al. 1996). Our finding that these gene products are required for normal microtubule stability in vivo is consistent with the role defined in vitro. The phenotypes of cells deficient for Cinlp or Pac2p are similar to cells with lowered tubulin activity. Loss of the Cin/Pac2p pathway is not lethal for S. cermisiaecells, however, indicating that some functional tubulin is still produced in its absence. Other cellular mechanisms must exist that lead to the generation of properly folded tubulin. This could be accomplished by an unknown activity or perhaps solely by the folding abilities intrinsic to the tubulin polypeptides.

The first step in the mammalian @-tubulin folding pathway defined by in vitro studies is performed by a cytoplasmic chaperonin complex (c-cpn) (GAO et al. 1993; TIAN et al. 1996). c-cpn also folds actin into a form competent for assembly into filaments (GAO et al. 1992; CHEN et al. 1994; VINH and DRUBIN 1994). In contrast, the @-tubulin released by c-cpn is not assembly competent, but requires the sequential actions of cofactors D (similar to Cinlp), E (similar to Pac2p) and C to achieve this form (TIAN et al. 1996). Consistent with this finding, we observed no defect in actin distribution in cinlA or pac2A cells (unpublished observations). The final step in the in vitro @-tubulin folding pathway is performed by cofactor C, which has no close sequence homologue encoded by the S. cerevisiae genome. Either the cofactor C step is not essential for the S. cerevisiae pathway, or it is performed by a factor with a different primary amino acid sequence.

The in vitro tubulin folding experiments did not define roles for Cin2p and Cin4p. There are now multiple lines of genetic evidence linking the functions of Cin2p and Cin4p with those of Cinlp and Pac2p: (1) defects affecting each cause an array of similar phenotypes; (2) multiple mutants are viable and no more sensitive to benomyl than any single mutant; (3) extra copies of CZNl could suppress loss of the other three, but not other mutations that affect tubulin function and (4) CINl mutations could suppress cin4-4 in an allele-specific manner. Two additional observations link the functions of Cin2p and Cin4p: (1) cin2A cin4A double mutants display the less sensitive to benomyl phenotype characteristic of cin4A (STEARNS et al. 1990) and (2) Cin2p was found to interact with Cin4p in a “two-hy- brid” interaction genetic screen (B. FEIERBACH and T. STFARNS, personal communication). Since mammalian homologues of Cin2p and Cin4p are apparently not required for the in vitro folding of @-tubulin, it is possible that (if they exist) they act upstream of cofactor D/ Cinlp in vivo. An attractive hypothesis is that they act to regulate the @-tubulin folding activity of cofactor D/

Cinlp. This view, supported by the findings reported here, is colored by our knowledge that Cin4p is a guanine nucleotide-binding protein, a class of proteins that perform regulatory roles in numerous cellular processes (BOURNE 1995; NEER 1995).

Extra copies of CINl were found to suppress the benomyl sensitivity of cin2A, cin4A and pac2A but not tubl-1 or tub3A. The suppression was therefore not caused by a mechanism that nonspecifically increased benomyl resistance. Rather, it must reflect some aspect of the mechanism of this microtubule-stabilizing pathway. Most reasonably, Cinlp must perform a critical role in this pathway and act closest to the microtubule target. This view is consistent with the failure to observe a requirement for mammalian forms of Cin2p and Cin4p in vitro, but does not account for the in vitro requirement for cofactor E (Pac2p). Resolution of this inconsistency will require further study. Upstream roles for Cin2p and Cin4p are also supported by the previously reported epistasis relationship (STEARNS et al. 1990) and, as discussed below, our finding of a unique genetic interaction between CZN4 and CZNl.

Four CINl alleles were identified that could dramati- cally raise the benomyl resistance of cin4-4 but had no effect upon other cin4 alleles, as well as cin2A and pac2A. Such allele-specific suppression can reflect changes affecting the physical interaction between two gene products (BOTSTEIN and MAURER 1982). An alter- native explanation for our observed allele specificity is that cin4-4 encodes the only tested form of Cin4p that is suppressible. In either case, our findings indicate a close relationship between Cinlp and Cin4p. An essential property of many guanine nucleotide-binding proteins is their ability to exist in two states: either GTP bound or GDP bound. These two states often confer different regulatory influences on their targets of actions. The cin4-4 allele causes a greater sensitivity to benomyl than cin4A and is altered in a residue conserved among guanine nucleotide-binding sites (T. STEARNS, C. CLARK, M. A. Hour, R. KAHN and D. BOTSTEIN, unpublished data). It seems possible that the cin4-4 defect creates a form that is locked into a state that is inhibitory for Cinlp function. The clustered CZNl alleles that overcome this inhibition might therefore define a site that participates in the Cinlp-Cin4p interaction. If the Cinlp changes create a form that is now resistant to the inhibitory affects of Cin44p, we would expect the suppression yielded to be dominant. Dominance for suppression was observed for all four CZNl-440 mutants.

It seems most likely that we have demonstrated the in vivo consequences for loss of the @-tubulin folding pathway defined in vitro. We can speculate that the functions of Cin2p and Cin4p are to regulate the functions of the folding activities, cofactor D/Cinlp and cofactor E/Pac2p. Perhaps the cellular requirement for tubulin

Microtubule-Stabilizing Pathway 857

subunits is transmitted via Cin2p and Cin4p to the tubulin folders, which also may act as or interact with cellular tubulin stores (see TIAN et al. 1996). In nitro studies of the effects of Cin2p and Cin4p will be required to address these issues.

The authors thank LAURA TOTIS and NELSON COLE for technical assistance and FRANK COTTINGHAM, KATIE FARR and ERIC SCHOTT for helpful comments on the manuscript. This study was supported by National Institutes of Health grant GM40714 awarded to M.A.H.

LITERATURE CITED

BOTSTEIN, D., and R. MAURER, 1982 Genetic approaches to the analysis of microbial development. Annu. Rev. Genet. 16: 61-83.

BOTSTEIN, D., N. SEGEV, T. STEARNS, M. A. Hour, J. HOLDEN et al., 1988 Diverse biological functions of small GTP-binding proteins in yeast. Cold Spring Harbor Symp. Quant. Biol. 53: 629- 636.

BOURNE, H. R., 1995 GTPases: a family of molecular switches and clocks. Philos. Trans. R. SOC. Lond. Biol. Sci. 349: 283-289.

CHEN, X., D. S. SLJLI.IVAN and T. C. HUFFAKER, 1994 Two yeast genes with similarity to TCP-1 are required for microtubule and actin function in vivo. Proc. Natl. Acad. Sci. USA 91: 9111-9115.

GAO, Y., J. 0. THOMAS, R. L. CHOW, G. LEE and N. J. COWAN, 1992 A cytoplasmic chaperonin that catalyzes @-actin folding. Cell 69:

GAO, Y., I. E. VAINBERG, R. L. CHOW and N. J. COWAN, 1993 Two cofactors and cytoplasmic chaperonin are required for the folding of cy- and @-tubulin. Mol. Cell. Biol. 13: 2478-2485.

GEISER, J. R., E. J . SCHOTT, N. B. COLE, G. BHATTACHARYYA, L. J. TOTIS et al., 1997 S. cereuisiae genes required in the absence of the CZN8-encoded spindle motor act in functionally diverse mitotic pathways. Mol. Biol. Cell (in press).

Hour, M. A., T. STEARNS and D. BOTSTEIN, 1990 Chromosome insta- bility mutants of Saccharomyces cereuisiae that are defective in microtubule-mediated processes. Mol. Cell. Biol. 10: 223-234.

HUFFAKER, T. C., J. H. THOMAS and D. BOTSTEIN, 1988 Diverse ef-

1043-1050.

fects of @-tubulin mutations on microtubule formation and function. J. Cell Biol. 106: 1997-2010.

JACOBS, C. W., A. E. M. ADAMS, P. J. SZANISZLO and J. R. PRINGLE, 1988

J. Cell Biol. 107: 1409-1426. Functions of microtubules in the Saccharomyces cermisiaecell cycle.

NEER, E.J., 1995 Heterotrimeric G proteins: organizers of trans- membrane signals. Cell 80: 249-257.

NEFF, N. F., J. H. THOMAS, P. GRISAFI and D. BOTSTEIN, 1983 Isola- tion of the &tubulin gene from yeast and demonstration of its essential function in vivo. Cell 33: 211-219.

RIEHEMANN, IC, and C. SORG, 1993 Sequence homologies between four cytoskeleton-associated proteins. Trends Biochem. Sci. 18:

ROTHSTEIN, R., 1991 Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzy- mol. 194 281-301.

SCHATZ, P. J., L. PILIJJS, P. GRISAFI, F. SOLOMON and D. BOTSTEIN, 1986a Two functional cy-tubulin genes of the yeast Saccharomyces cereuisiae encode divergent proteins. Mol. Cell. Biol. 6: 3711- 3721.

SCHATZ, P. J., F. SOLOMON and D. BOTSTEIN, 1986b Genetically essential and nonessential cy-tubulin genes specify functionally interchangeable proteins. Mol. Cell. Biol. 6: 3722-3733.

SHERMAN, F., G. R. FINK and J. B. HICKS, 1983 Methods in Yeast Genet- ics. Cold Spring Harbor Press, Cold Spring Harbor, NY.

STEARNS, T., and D. BOTSTEIN, 1988 Unlinked noncomplementation: isolation of new conditional-lethal mutations in each of the tubulin genes of Saccharomyces cereuisim. Genetics 119: 249-260.

STFARNS, T., M. A. HOW and D. BOTSTEIN, 1990 Yeast mutants sensitive to antimicrotubule drugs define three genes that affect microtubule function. Genetics 124: 251-262.

THOMAS, J. T., N. F. NEFF and D. BOTSTEIN, 1985 Isolation and characterization of mutations in the @-tubulin gene of Saccharomyces cereuisim. Genetics 111: 715-734.

TIAN, G., Y. HUANG, H. ROMMEIAERE, J. VANDEKERCKHOVE, C. AMPE et al., 1996 Pathway leading to correctly folded @-tubulin. Cell

VINH, D. B., and D. G. DRUBIN, 1994 A yeast TCP-1-like protein is required for actin function in vivo. Proc. Natl. Acad. Sci. USA 91: 9116-9120.

82-83.

86: 287-296.

Communicating editor: M. D. ROSE

saccharomyces cerevisiae pac2 functions with cznl, 2 and 4 ... · saccharomyces cerevisiae pac2...

Documents