The identification of a Cdc31p-interacting kinase, Kic1p, marks the first report of a centrin associated with a protein kinase. In addition, this is the first report of a non-SPB (centrosome) function for a centrin protein. We demonstrate that Kic1p's kinase activity is dependent upon Cdc31p and that the kinase domain is required for proper cell wall assembly and cell integrity. Examination of four temperature-sensitive cdc31 alleles, previously characterized as defective for SPB duplication, revealed that these mutants exhibited allele-specific cell integrity and morphology defects. We conclude that that Cdc31p is critically involved in Kic1p's function in cell integrity and morphogenesis, but that Kic1p is not required for Cdc31p's SPB duplication function. Consistent with this, the combination of cdc31-1 and kic1-1, two alleles that do not generate lysis on their own, resulted in a severe synthetic lysis defect, similar to the most severe kic1 mutation. The simplest interpretation of these results is that Kic1p and Cdc31p interact, in vivo, to regulate some aspect of cell wall assembly.
The identification of allele-specific cell integrity and morphology defects for different cdc31
alleles was surprising. The cdc31-2
, and CDC31-16
mutants had lysis defects of varying severity, and the cdc31-1
mutants had elongated bud growth. The variation in cdc31
phenotypes suggests that CDC31
might be required for more than one morphological process. One possibility is that, like calmodulin, Cdc31p may interact with several different proteins with distinct functions. Different mutations may have more or less severe defects depending upon the specific interactions that are most affected. It is intriguing that cdc31-1
maps between the two NH2
-terminal EF-hands (A48T), cdc31-5
maps to the central α-helical domain (P94S), and both cdc31-2
map between the two COOH-terminal EF-hands (D131N and E133K, respectively; Vallen et al., 1994
). Given the location of the mutations, it is tempting to speculate that the central domain is important for cell integrity, whereas the NH2
-terminal half of the protein preferentially effects bud morphology. Whereas cdc31-1
does not exhibit a lysis defect on its own, the synthetic lysis defect observed for the cdc31-1 kic1-1
double mutant suggests that this mutation may partially compromise the activity of Kic1p or the interaction between Kic1p and Cdc31p.
In contrast to the effects of loss of function mutations, when wild-type Kic1p was overexpressed it caused an elongated cell phenotype (Fig. B), similar to that observed for cdc31-1 and cdc31-2. Interestingly, co-overexpression of Cdc31p caused cells to regain their normal rounded morphology. If the elongated cell phenotype were simply due to overexpression of Kic1p kinase activity, which is Cdc31p dependent, we would expect that co-overexpression of Cdc31p would increase kinase activity and further exacerbate the elongated cell phenotype. However, we found the opposite to be true. We observed suppression of the elongated cell phenotype, and therefore the phenotype appears to be independent of Kic1p kinase activity. Indeed the elongated cell phenotype was also observed when kic1-1 (a kinase down mutant) was overexpressed (data not shown). We propose that overexpression of Kic1p titrates Cdc31p away from another protein(s) whose interaction with Cdc31p is required for normal cell morphology. However, we cannot rule out the possibility that overexpression of the non-kinase domain of Kic1p generates the elongated cell phenotype.
Our results complement recent unpublished data from the laboratory of F. Klis (Vossen, J., and F. Klis, personal communication). These researchers independently identified KIC1 on the basis of the sensitivity of the mutant to calcofluor white. They have also found that kic1 mutants are highly fragile and defective in cell separation. The mutants were resistant to K1 killer toxin, because of reduced β1-6 glucan in their cell walls. They also found that the cell walls were resistant to Zymolyase, a β1-3 glucanase, indicting that the outer protein or mannan layer was less permeable than in wild-type cells. These results are consistent with our electron microscopy observations that show that kic1 cell walls are disorganized, lack a wild-type glucan layer, and have increased levels of electron-dense mannans. In addition, these researchers have shown that loss of Kic1p function upregulates the HOG pathway (high osmolarity/glycerol). When the HOG pathway is upregulated, cells produce more of the EXG1 exoglucanase with reduces β1-6 glucan levels. In addition, upregulation of the HOG pathway triggers accumulation of glycerol in cell increasing their osmotic potentials. This could explain part of the lysis defect of kic1 mutants. Increased osmotic potential would draw more water into the cells creating additional pressure on the cell walls leading to lysis.
mutants lyse near the bud neck suggesting that they are lysing either at the neck, or at the bud or birth scars. Mutations in chitin deposition in the cell wall often lead to lysis at bud or birth scars which are largely composed of chitin (for review see Cid et al., 1995
). Our electron microscopic analysis of the kic1
mutants identified aberrant electron transparent structures, presumably comprised of chitin, at the bud neck. Mutants sometimes had what appeared to be either multiple primary septa or chitin rings. These structures often appeared frayed and looked like weak spots in the wall. It is important to note that the kic1
defect is not the same as that caused by mutations in PKC1
, the gene for protein kinase C (Levin and Bartlett-Heubusch, 1992
; for review see Cid et al., 1995
). Like kic1
mutants exhibit lysis that is suppressed by the addition of 1 M sorbitol to the media. However, unlike kic1-1
mutants lyse at the tips of small buds, where there is a noticeable thinning of the cell wall. Furthermore, pkc1
mutants arrest at the G2
–M transition with duplicated DNA content and small buds, whereas kic1
mutants do not arrest at the G2
–M transition and complete multiple rounds of the cell cycle. Accordingly, the kic1
mutants accumulate fewer small-budded cells and the lysis defect is not always suppressed by sorbitol. The kic1
mutants also exhibit an abnormal bud neck phenotype that is not seen in pkc1
mutants. Therefore we propose that Kic1p and Pkc1p act in separate morphogenic pathways.
Kic1p is a member of a sub-group of PAK1/Ste20p-like kinases that have NH2
-terminal kinase domains and COOH- terminal regulatory regions. Unlike other members of the PAK1/Ste20p family, this subfamily lacks Cdc42- and Rac-binding domains. The demonstration that the kinase activity of Kic1p is Cdc31p dependent raises the possibility that centrin may regulate the activity of NH2
-terminal Ste20p-like kinases in other organisms just as Cdc42p/Rac regulates the function of the COOH-terminal kinases. Interestingly, Kic1p and a subset of PAK kinases contain an eleven amino acid motif (AKPXSILXD/ELI) that is found just COOH-terminal to the kinase domain (Fig. B
). It has recently been reported that this sequence regulates the binding of G protein β-subunits to PAK/Ste20 kinases (Leeuw et al., 1998
). This domain partially overlaps the putative Cdc31p-binding domain of Kic1p (Vallen et al., 1994
; Spang et al., 1995
). The G protein β-subunit that binds to Ste20p was Ste4p, a protein of 46 kD. It is tempting to speculate that the 40-kD protein we see associated with Kic1p might be a β-subunit that co-operatively or competitively regulates Kic1p function with Cdc31p.
The class B and C mutants (Table ) also exhibited mild spindle migration defects, which resulted in small-budded binucleate cells. These may be due to the mislocalization of actin during the stages of bud morphogenesis. Previous work has documented that spindle migration and orientation in yeast requires both functional astral microtubules (Palmer et al., 1992
; Sullivan and Huffaker, 1992
) and a polarized actin cytoskeleton (Palmer et al., 1992
; Wang and Bretscher, 1995
). In the absence of any obvious defects in microtubule structure (data not shown), it is reasonable to suggest that the kic1
binucleate phenotype is due primarily to actin defects. The wide bud neck phenotype could also be caused by the observed actin defects. In many cases actin was found at the bud site and neck after bud emergence had progressed significantly. This is not seen in wild-type cells. Since secretion is actin directed this might lead to increased deposition of cell wall materials. In this respect it is important to note that some PAK kinases have been implicated in regulating actin organization.
Cdc31p is involved in both SPB duplication and cell integrity/morphogenesis. Is this a coincidence or are they coordinated in some way? The earliest step in SPB duplication, the appearance of the SPB satellite body, occurs after telophase but before START. Bud growth is initiated at START and must be completed for proper completion of both the nuclear and cell division pathways. The involvement of Cdc31p in two disparate processes raises the possibility that Cdc31p plays an explicit role in the coordination of these two major cell cycle processes.