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© The Rockefeller University Press,
0021-9525/1998//873 $5.00
The Journal of Cell Biology, Volume 140, Number 4,
, 1998 873-883
Article |
Smy1p, a Kinesin-related Protein That Does Not Require Microtubules
Abstract. We have previously reported that a defect in Myo2p, a myosin in budding yeast (Saccharomyces cerevisiae), can be partially corrected by overexpression of Smy1p, which is by sequence a kinesin-related protein (Lillie, S.H., and S.S. Brown. 1992. Nature. 356:358– 361). Such a functional link between putative actin- and microtubule-based motors is surprising, so here we have tested the prediction that Smy1p indeed acts as a microtubule-based motor. Unexpectedly, we found that abolition of microtubules by nocodazole does not interfere with the ability of Smy1p to correct the mutant Myo2p defect, nor does it interfere with the ability of Smy1p to localize properly. In addition, other perturbations of microtubules, such as treatment with benomyl or introduction of tubulin mutations, do not exacerbate the Myo2p defect. Furthermore, a mutation in SMY1 strongly predicted to destroy motor activity does not destroy Smy1p function. We have also observed a genetic interaction between SMY1 and two of the late SEC mutations, sec2 and sec4. This indicates that Smy1p can play a role even when Myo2p is wild type, and that Smy1p acts at a specific step of the late secretory pathway. We conclude that Smy1p does not act as a microtubule-based motor to localize properly or to compensate for defective Myo2p, but that it must instead act in some novel way.
IN SACCHAROMYCES CEREVISIAE, virtually all growth occurs in the bud rather than in the mother cell. MYO2 encodes an unconventional class V myosin that has been implicated in this polarized growth by studies of the temperature-sensitive myo2-66 mutant. This mutant fails to target growth to the bud at restrictive temperature, resulting in abnormally large mother cells (see Fig. 1 a; Johnston et al., 1991). Because vesicles were observed to accumulate in the mutant, Johnston et al. (1991) proposed that Myo2p targeted growth by delivering secretory vesicles to the bud. The recent finding that in chick brain a class V myosin is associated with synaptic vesicles (Prekeris and Terrian, 1997) would seem to suggest that other class V myosins might have similar functions. The evidence that Myo2p is a secretory vesicle motor, however, is far from conclusive (Liu and Bretscher, 1992; Govindan et al., 1995), and there are other ways that Myo2p might target growth. For example, it might deliver elements of the ER or Golgi apparatus, which are found in proximity to the bud at a very early stage of bud growth (Preuss et al., 1992). Another possibility is that Myo2p might reside at the bud tip as part of a complex that anchors the actin cytoskeleton or cell wall–synthesizing machinery.
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(SMY1 deletion) double mutant is dead at permissive temperature, despite the fact that each single mutant appears wild type under the same conditions (Lillie and Brown, 1992). This demonstrates that Smy1p is not simply providing some "spillover function" when overexpressed, but also that it is essential in a myo2 mutant background. An even stronger indication of the functional significance of the interaction is that Myo2p and Smy1p colocalize, and that their localizations respond identically to several cell perturbations (Lillie and Brown, 1994). Furthermore, overexpression of Smy1p not only restores Myo2p mutant localization, but it also enhances the localization of wild-type Myo2p. What is particularly surprising about the close relationship between Myo2p and Smy1p is that although both are putative motor proteins, they are predicted to interact with different cytoskeletal filaments. Myo2p is expected to interact with actin filaments, which have also been implicated in polarized growth by their changing localization during the cell cycle (Adams and Pringle, 1984; Kilmartin and Adams, 1984) and by studies of actin mutants (Novick and Botstein, 1985). Smy1p, on the other hand, is by sequence a kinesin-related protein (Lillie and Brown, 1992) and is thus expected to interact with microtubules. Some time ago, it was believed that cytoplasmic microtubules would be required for bud growth, given their location; they emanate from the spindle pole body at the nuclear membrane and extend into the growing bud. Genetic and nocodazole studies have clearly shown, however, that microtubules are not required for bud growth, but instead, that they are required for nuclear migration and mitosis (Jacobs et al., 1988; Huffaker et al., 1988). Although microtubules together with Smy1p might act as a backup system that is needed for bud growth only when Myo2p is defective, such functional overlap would nonetheless be surprising.
Another consideration is the relative divergence of Smy1p compared to other members of the kinesin superfamily (Lillie and Brown, 1992; Goldstein, 1993), which raises the question of whether it is truly a microtubule-based motor. The three-dimensional structures of the motor domains of both myosin and kinesin have been determined (Rayment et al., 1993; Kull et al., 1996). Whereas these two families of motors show a remarkable similarity in their core structures, the insertions that confer microtubule vs. myosin binding are quite different (Kull et al., 1996; Woehlke et al., 1997). Smy1p has some sequence divergence in regions corresponding to the microtubule-binding regions, but not enough to judge whether it is likely to bind microtubules. For example, in the loop determined by mutagenesis studies to be the most critical for microtubule binding (Woehlke et al., 1997), Smy1p retains an important arginine (corresponding to R278) but has changes in other conserved residues. On the other hand, costal2, another divergent kinesin-related protein, does not retain this arginine but does bind microtubules (Sisson et al., 1997). Smy1p also has changes that could affect other aspects of motor function. It is divergent at two otherwise extremely conserved positions (corresponding to E250 and E311 in Woehlke et al., 1997) where mutations affect the ability of kinesin to be activated by microtubule binding. Other sites of divergence might affect interactions with nucleotide (N-1 through N-4 in Sablin et al., 1996); costal2 shows even more divergence in these regions (Sisson et al., 1997). In summary, sequence comparison does not allow us to predict whether Smy1p can bind microtubules, but it does raise the possibility that Smy1p, like costal2, may be a poor or even nonfunctional motor.
In this report, we have looked for functional evidence to determine whether Smy1p interacts with microtubules. Our results show that Smy1p does not require microtubules to localize properly or to compensate for mutant Myo2p defects.
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Materials and Methods |
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To alter amino acid 119 of Smy1p from Gly to Glu, a missense mutation (smy1-3) was introduced by site-directed mutagenesis (using the double-primer method; Sambrook et al., 1989) of a single-stranded template, using the following mutagenic primer: GTTTCAGCGAAAAGTCCTA. The mutation was confirmed by sequencing and then shuttled as a KpnI-XbaI fragment to KpnI-XbaI-cut pBR-SMY1 [pBR322 (Sambrook et al., 1989) with a 
4.5-kb EcoRV SMY1-containing insert]. The 3.3-kb HpaI-EcoRV smy1-3–containing fragment from this plasmid was inserted into SmaI-digested YEp352 (Hill et al., 1986) to give YEpsmy1-52. YEpSMY1-52 was constructed by shuttling the corresponding wild-type SMY1 segment from pBR-SMY1 to YEp352.
Nocodazole Treatment and Immunofluorescence
Nocodazole (Sigma Chemical Co., St. Louis, MO) was added to exponentially growing cultures (1–2 x 106 cells/ml) as described by Jacobs et al. (1988), except that stocks were stored in aliquots at –80°C, and these were warmed briefly to 50°C immediately before use to ensure complete solubilization. At each time point, samples were fixed, and an aliquot was processed for Smy1p, Myo2p, or microtubule staining by indirect immunofluorescence, as described by Lillie and Brown (1994). (Formaldehyde was added directly to shaking cultures to minimize loss of labile structures.) Samples were examined using a range of antitubulin concentrations to optimize conditions for detection of microtubules.
Cells eventually escape the effects of nocodazole (Jacobs et al., 1988), and this occurs more rapidly when a greater than optimum concentration is used (see Fig. 2, compare a and d). Therefore, for each strain and set of experimental conditions, we carried out pilot experiments using a series of nocodazole concentrations to find the narrow window where the drug had its greatest effect. The length of the nocodazole block is also dependent on a number of other factors. Haploids are more resistant to the drug than diploids (our observations and Stearns, T., personal communication), and nocodazole is more effective in rich medium than in selective medium. Furthermore, escape is enhanced at a higher temperature. We took advantage of these findings to maximize the length of the nocodazole block. Diploids were used in nocodazole experiments, cells were shifted to rich medium if they had been grown in selective medium, and the experiment in Fig. 2 was carried out without elevating the temperature. In addition, for the experiment in Fig. 1, we used diploids heterozygous for the tub1-1 allele. This 
-tubulin mutation increases sensitivity to nocodazole, even when heterozygous (our observations and Stearns, T., personal communication), which is useful, because the homozygous but not the heterozygous tub1 mutant shows an elevated number of large-budded cells even at permissive temperature in the absence of nocodazole. We confirmed that tub1 does not qualitatively alter the results in experiments using other myo2 strains that were wild type for TUB1 (not shown).
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The experiment above has a couple of complications, which, however, do not abrogate our conclusion. The first is that the shift to restrictive temperature causes a transient increase in the fraction of unbudded cells, peaking at 1 h (i.e., at 0 h in Fig. 1 c for all four cultures). This has been observed previously with wild-type cells (Johnston and Singer, 1980; Plesset et al., 1987), and it is temporally correlated with transient disorganization of the actin cytoskeleton (Lillie and Brown, 1994). The cultures did not return to the initial percentage of unbudded cells seen at room temperature, but they instead reached a new, higher plateau after temperature shift (compare open symbols at –1 h and >2 h in Fig. 1 c). The second complication is that yeast eventually escapes the effects of nocodazole (Jacobs et al., 1988). We used a number of strategies to maximize the length of the nocodazole block (refer to Materials and Methods), but were only able to prolong it long enough to allow about half the cells to reach the large-budded arrest point. Microtubule staining began to reappear at 2 1/2 h of nocodazole treatment in a small fraction of the cells. Concomitant with the reappearance of microtubules, the proportion of large-budded cells began to decline (Fig. 1 b, closed circles) as the proportion of unbudded cells began to increase (Fig. 1 c, closed circles), an indication that cells were indeed resuming mitosis.
The most effective way we found to prolong the nocodazole block was to lower the temperature. Therefore, to confirm and extend the results obtained at restrictive temperature, we performed an experiment at room temperature. A change in strains and protocol from that of Fig. 1 was necessitated by the fact that the myo2 mutant does not require multicopy SMY1 at room [permissive] temperature. Instead, we compared the effects of nocodazole on closely matched homozygous myo2 and wild-type diploids (made by mating sister segregants obtained after several backcrosses). Since the myo2 smy1 double mutant is inviable (Lillie and Brown, 1992), we infer that nocodazole should have an effect on the rate of bud growth if microtubules were required for the compensating effect of Smy1p in the myo2 mutant at nominally permissive temperature. In this experiment, cytoplasmic microtubules were no longer detectable by 30 min after nocodazole addition and started to reappear only at 63/4 h. Most (see below) myo2-mutant cells and virtually all wild-type cells were able to reach large-budded arrest by 21/2 h with approximately the same kinetics (Fig. 2 a). No additional increase in the fraction of large-budded cells was seen in the next 4 h. Furthermore, when either a lower (not shown) or higher (Fig. 2 d) dose of nocodazole was used, both of which imposed a shorter block, the myo2-mutant strains once again displayed similar rates of bud growth and escape from nocodazole as the wild-type strains did.
We conclude that the room temperature experiment does indeed confirm the results obtained in the experiment performed at restrictive temperature in that a large fraction of mutant cells behave like wild-type cells in response to nocodazole. We were, however, puzzled by the difference in plateau values for wild-type vs. mutant cells. The mutant cells that failed to reach the large-budded stage did not appear to arrest at a specific stage because both unbudded and small-budded cells remained (Fig. 2, b and c). It seemed unlikely that mutant cells were more susceptible to some poisoning effect of nocodazole because the same fraction of myo2 cells failed to reach the large-budded stage using a range of nocodazole concentrations (from 5 to 15 µg/ml; compare Fig. 2, a and d). We therefore examined cells that had not been treated with nocodazole for their ability to grow. The same strains that we used in Fig. 2 were plated as single cells at room temperature (in the absence of nocodazole) and examined microscopically at various times thereafter. Essentially all of the wild-type control cells and most of the mutant cells were able to grow buds and divide several times during the timecourse of the experiment (Fig. 3). However, 10–20% of the myo2-mutant cells never divided; instead, they remained as single cells that were either unbudded or had buds of various sizes. These nondividing myo2 cells were not dead by the criterion of dye exclusion (not shown). Although we cannot explain this behavior, it could account for the difference in plateau values between wild-type and myo2-mutant cells in Fig. 2.
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- or β-tubulin mutations (a total of eight alleles from the three tub genes were tested; Table II) despite the fact that myo2 is synthetically lethal with smy1 (Lillie and Brown, 1992). Furthermore, we found that myo2- mutant strains were not more sensitive than related wild-type strains to benomyl, another drug that acts on microtubules (Stearns and Botstein, 1988). Tetrads of four well-backcrossed myo2/MYO2 diploids were dissected, and segregants were replica-pronged onto YPD plates containing 0, 10, 15, 20, and 25 µg/ml benomyl (Thomas et al., 1985) at 22°C. For one of the crosses, cells were spread on sectors of the same plates to better assess relative growth. No consistent differences between myo2 and wild-type segregants were seen. Both grew well at 10 µg/ml of benomyl, less well at 15 µg/ml, and very little growth was seen at 20 or 25 µg/ml. These results are consistent with the hypothesis that Smy1p does not require microtubules to compensate for the Myo2p defect.
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background to avoid staining of wild-type protein, and was indistinguishable from that of wild-type Smy1p (data not shown). Furthermore, the mutation did not interfere with the ability of overexpressed Smy1p to overcome the defects of the myo2 mutant. A myo2 mutant strain (SLY34) carrying multicopy mutated smy1-3 (YEpsmy1-52) grew as well as the same strain with wild-type SMY1 (YEpSMY1-52) when incubated for 4 d on a YPD plate at restrictive temperature (31°C). Subtle differences were sometimes seen; these may be caused by a slight loss of stability of the mutant protein at elevated temperature, given the lack of such ephemeral differences in the experiment below. In contrast, no growth was seen with the control plasmid (Yep352), and all strains grew well on a YPD plate at permissive temperature. A reservation is that the myo2 mutant has an endogenous wild-type SMY1 gene in addition to the overexpressed smy1-3 gene. This could complicate interpretation if wild-type Smy1p formed functional heterodimers with mutant Smy1p (or if there were an unexpectedly high rate of gene conversion). Therefore, we used a second functional test that avoided these complications, asking whether smy1-3 prevented lethality (Lillie and Brown, 1992) in a myo2 smy1 double mutant. A heterozygous diploid strain (SLY103) carrying each of the same plasmids as described above was subjected to tetrad analysis at room temperature. We found that in the vast majority of tetrads (those that had retained plasmid) all segregants were viable, except that as expected (Lillie and Brown, 1992), no live double-mutant segregants were recovered from SLY103-carrying control vector. Thus, smy1-3 was able to prevent lethality in the double mutant. Furthermore, the growth rates of double-mutant segregants carrying wild-type versus mutant SMY1 (strains SLY191, SLY192, SLY194, and SLY195) incubated at 25°C in YPD were indistinguishable when followed by OD over a period of several days (with dilutions to maintain cells in log phase). Therefore, smy1-3 not only prevented lethality, but it also allowed cells to grow as well as they did with SMY1. We conclude from the above experiments that Smy1p does not need motor activity to localize properly and to compensate for Myo2p defects. The above-mentioned kar3-1 mutation causes Kar3p localization to microtubules to become detectable (Meluh and Rose, 1990). An attractive interpretation is that the mutant Kar3p cannot bind ATP well and is therefore blocked at a step of the ATPase cycle when it is tightly bound to microtubules (see Hackney, 1994). Therefore, we looked carefully for mutant Smy1p localization to microtubules but were unable to detect any. kar3-1 has dominant-negative effects, perhaps because tightly bound mutant protein competes with other microtubule interactions. We looked for dominant effects with smy1-3 but found no evidence of this. These findings are consistent with the evidence presented above that Smy1p is not a microtubule-based motor.
A deletion of amino acids 20–137 (roughly the first third of the Smy1p "motor" domain including the P loop) abolishes localization and the ability to correct myo2-mutant defects without reducing the amount of Smy1p present (as judged by Western analysis; data not shown). This is not conclusive proof that the motor domain of Smy1p is functionally important because the deletion might cause folding problems that interfere with other domains. However, a similar deletion in kinesin did not abolish microtubule binding (Yang et al., 1989), suggesting that our deletion may not have such global effects. Assuming that the motor domain is indeed important, it seems likely that this domain is involved in a novel function that Smy1p has acquired.
Synthetic Lethal Relationships
Although they should not be overinterpreted, synthetic interactions (i.e., a more than additive phenotype in a double mutant) can provide valuable clues about function. We have used this approach to ask several questions about Smy1p. A summary of crosses performed with smy1 (and myo2) mutants is provided in Table II. First, we looked for synthetic interactions between smy1 and deletions of other kinesin-related genes. We reasoned that Smy1p might indeed be a microtubule-based motor, even though this activity is not needed for localization or to compensate for the myo2 mutation. If so, the lack of a phenotype upon SMY1 deletion might indicate redundancy with another kinesin-related protein. However, we found no indication of a synthetic interaction between smy1 and deletions in KAR3, KIP1, KIP2, or CIN8. The completion of the yeast genome project has revealed a sixth kinesin- related gene in yeast, KIP3, which also does not have a synthetic interaction with smy1 (DeZwaan et al., 1997; Hoyt, M.A., personal communication). The only other microtubule-based motor in yeast is dynein, and there is no synthetic interaction between smy1 and deletion of DYN1 (Cottingham and Hoyt, 1997). Synthetic interactions have implicated all of these microtubule-based motor proteins in partially overlapping aspects of nuclear migration and mitosis (Cottingham and Hoyt, 1997; DeZwaan et al., 1997). SMY1 stands alone in its failure to show any synthetic interactions.
We have also used this approach to look for further clues as to the nature of Smy1p's relationship with Myo2p and/or other components of the actin cytoskeleton (Table II). No genetic interactions were seen, except for a subtle interaction between smy1 and deletion of the gene encoding the putative conventional myosin (MYO1). We also detected a subtle interaction between the MYO1 deletion and myo2, and we are investigating these interactions further.
The most informative results we obtained were with mutants defective in the secretory pathway (SEC mutants). Govindan et al. (1995) have shown that myo2 is synthetically lethal with mutations in most of the late (but not the early) SEC genes, suggesting that Myo2p plays a role at a late stage of secretion. When we tested smy1 against the same alleles, we found synthetic lethality with two of these late SEC mutations (sec2 and sec4), but not with the others nor with the early SEC mutations we tested. These results are interesting for two reasons. First, they show that Smy1p plays a role in exocytosis, even when Myo2p is wild type. In other words, they rule out the possibility that the only function of Smy1p is to stabilize a mutant Myo2p. Second, the fact that Smy1p interacts (genetically) with only a subset of the proteins that interact with Myo2p may well be a significant clue to the role it plays in secretion.
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Our conclusion that microtubules are not required for Smy1p localization and function depends on knowing that nocodazole has truly abolished all microtubules. Although we have been careful to stipulate that our results were obtained in the absence of "detectable" microtubules, we are confident that cytoplasmic microtubules were completely abolished for several reasons. First, there are not many cytoplasmic microtubules in yeast (an average of three per cell), and cytoplasmic microtubule staining corresponds to individual microtubules (Carminati and Stearns, 1997). Second, Jacobs et al. (1988) went to great lengths to demonstrate that nocodazole completely abolishes microtubules in S. cerevisiae. Third, microtubules were functionally absent; cells were unable to undergo mitosis. Furthermore, we did not begin to see escape from mitotic arrest until microtubules began to reappear. Thus, it is thoroughly unlikely that microtubules persisted that we failed to detect.
The fact that microtubules are not required for Smy1p to localize or to function led to the prediction that motor activity would not be required either. Studies of the smy1-3 mutation support this prediction. This mutation has no apparent effects on Smy1p, in contrast to the dramatic effects of the same mutation (kar3-1; Meluh and Rose, 1990) on another yeast kinesin-related protein, Kar3p. The loss of Kar3p function must be caused by a loss of motor activity because the mutation is in the P loop, a motif that interacts with the phosphate portion of nucleotide in many NTPases. The mutation alters a glycine that is absolutely conserved (Saraste et al., 1990) and whose alteration interferes with nucleotide binding and/or inactivates other NTPases (Clanton et al., 1987; Liu and Summers, 1988; Logan and Knight, 1993; Shen et al., 1994). Of particular relevance, given the structural similarity between kinesin and myosin (Kull et al., 1996), is the effect of this mutation on Dictyostelium myosin II (Gly184 to Glu); the mutant myosin no longer binds ATP, and actin binding becomes ATP insensitive (Ruppel, K., personal communication). This glycine is critical because of a structural constraint; any other amino acid in this position destroys the conformation of the P loop (Pai et al., 1990), which is essentially identical in all NTPases whose three-dimensional structures have been determined (Kull et al., 1996; Smith and Rayment, 1996). Thus, this mutation should have affected the ability of Smy1p to localize or to correct Myo2p defects if motor activity were required. (Note that this would be the prediction even if Smy1p were to travel along some novel substrate such as actin.) Our results do not rule out the possibility that Smy1p could be capable of motor activity, which is used for some unknown function that has not been revealed by phenotypic studies. However, our efforts to observe in vitro motility, ATP binding or hydrolysis, and cosedimentation with microtubules have all been negative. Furthermore, the divergence of Smy1p at many otherwise highly conserved positions (see Introduction) also suggests that it may not be a motor protein.
The synthetic lethal relationships that we have observed are consistent with earlier evidence that Smy1p is functionally linked with the actin rather than the microtubule cytoskeleton (Lillie and Brown, 1994). Of particular interest is the synthetic lethality observed between SMY1 deletion and mutations in two of the late SEC genes. The products of the late SEC genes function in the delivery or fusion of secretory vesicles with the cell membrane, a process in which actin (Novick and Botstein, 1985) and Myo2p (Govindan et al., 1995) have also been implicated. This synthetic lethality provides new clues about Smy1p function. The first is that Smy1p must have a function beyond that of stabilizing mutant Myo2p, since the synthetic lethality takes place in cells where MYO2 is wild type. Smy1p may nonetheless act via Myo2p; we have previously noted that SMY1 overexpression enhances the localization of even wild-type Myo2p (Lillie and Brown, 1994). The second clue is that smy1 is synthetically lethal with mutations in only two of the late SEC genes, whereas Govindan et al (1995) have shown that myo2 is synthetically lethal with most late SEC mutations. The two mutations with which smy1 is synthetically lethal, sec2 and sec4, are not simply the most deleterious of the late SEC mutations tested (Nair et al., 1990), thus ruling out the most trivial explanation of these results. Instead, recent studies indicate that the functions of Sec2p and Sec4p are intimately associated. Sec4p is a small GTPase (Salminen and Novick, 1987) that has been postulated to act as a conformational switch regulating either the delivery of vesicles or their subsequent capture at the site of fusion. Interestingly, Sec2p has been found to be an exchange factor for Sec4p (Walch-Solimena et al., 1997). Almost all of the other late SEC mutations we tested (sec3 [not shown], 5, 6, 8, 10, and 15), which are not synthetically lethal with smy1, are components of the "exocyst," a large complex of copurifying proteins (TerBush and Novick, 1995; TerBush et al., 1996). Sec4p continues to localize at the bud tip in exocyst mutants, indicating that the exocyst is not involved in vesicle localization, but rather, in some subsequent event (Walch-Solimena et al., 1997). Sec4p, however, does not localize in sec2 or myo2-66 mutants (Walch-Solimena et al., 1997). Taken together, these data indicate that Smy1p, Myo2p, Sec2p, and Sec4p are involved in event(s) that affect vesicle delivery and/or capture. Since the myo2 mutation is also synthetically lethal with most of the exocyst mutations, it may be sufficiently detrimental that it also affects subsequent events. We should point out that although the most obvious way for Myo2p to play a role is to be a secretory vesicle motor, there are a number of other possibilities (refer to Introduction).
In summary, Smy1p does not use microtubule-based motor activity to compensate for the mutant Myo2p defect. Regardless of whether or not it is capable of such an activity, Smy1p must function in some novel manner in its interaction with Myo2p. The family of actin-related proteins (Mullins et al., 1996) provides a precedent for proteins with structural similarity but divergent function; Smy1p may represent an example of such divergence in the kinesin superfamily.
Submitted:
We thank C. Schmidt (University of Michigan, Ann Arbor, MI) for in vitro motility assays; T. Huffaker for the gifts of benomyl and tub2 mutants, and for advice on the use of benomyl; A. Hoyt for tub1 and tub3 mutants; A. Hoyt and T. Stearns for advice on nocodazole; and B. Fuller (University of Michigan) for critical reading of the manuscript.
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