The yeast centrin, cdc31p, and the interacting protein kinase, Kic1p, are required for cell integrity - PubMed (original) (raw)

The yeast centrin, cdc31p, and the interacting protein kinase, Kic1p, are required for cell integrity

D S Sullivan et al. J Cell Biol. 1998.

Abstract

Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily. Previously centrins have been implicated only in microtubule-based processes. To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p). Kic1p is closely related to S. cerevisiae Ste20p and the p-21- activated kinases (PAKs) found in a wide variety of eukaryotic organisms. Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST-Kic1p and it bound to GST-Kic1p in gel overlay assays. Furthermore, GST-Kic1p exhibited in vitro kinase activity that was CDC31-dependent. Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity. We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects. The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect. We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro. We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.

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Figures

Figure 1

Figure 1

Kic1p kinase catalytic domain. (A) The Kic1p-kinase domain is aligned with its closest homologues: SOK1/Ysk1 (Pombo et al., 1996), CELT19A5 (Wilson et al., 1994), NIK1 (Su et al., 1997), MST1 (Creasy and Chernoff, 1995), PAK1 (Brown et al., 1996), and Ste20p (Leberer et al., 1992). The length in amino acids is indicated at the end of the bars. The percentage of identical residues with the Kic1p kinase domain is shown in the far right. (B) The NH2-terminal amino acids of Kic1p are aligned with the catalytic domains of MST1 from H. sapiens, PAK1 from H. sapiens, and Ste20p from S. cerevisiae. Identical residues are indicated by the black boxes and amino acid number is indicated on the right. The 11 subdomains conserved in all kinases are indicated (Hanks et al., 1988; Lindberg et al., 1992). A short domain of additional homology is marked by asterisks above and below the text.

Figure 1

Figure 1

Kic1p kinase catalytic domain. (A) The Kic1p-kinase domain is aligned with its closest homologues: SOK1/Ysk1 (Pombo et al., 1996), CELT19A5 (Wilson et al., 1994), NIK1 (Su et al., 1997), MST1 (Creasy and Chernoff, 1995), PAK1 (Brown et al., 1996), and Ste20p (Leberer et al., 1992). The length in amino acids is indicated at the end of the bars. The percentage of identical residues with the Kic1p kinase domain is shown in the far right. (B) The NH2-terminal amino acids of Kic1p are aligned with the catalytic domains of MST1 from H. sapiens, PAK1 from H. sapiens, and Ste20p from S. cerevisiae. Identical residues are indicated by the black boxes and amino acid number is indicated on the right. The 11 subdomains conserved in all kinases are indicated (Hanks et al., 1988; Lindberg et al., 1992). A short domain of additional homology is marked by asterisks above and below the text.

Figure 2

Figure 2

Cdc31p binds Kic1p directly and is part of a larger complex. (A) Cdc31p coprecipitates with Kic1p. GST, GST catalytic domain, and GST full-length Kic1p fusion proteins were purified from yeast (strains MY4124, MY4125, and MY4126, respectively) using glutathione–agarose under native conditions, separated on denaturing gels, and then Western blotted with anti-Cdc31p antibodies. Cdc31p band is indicated for part A and B. (B) Cdc31p interaction with Kic1p is calcium independent. The full-length Kic1p fusion protein was purified from MY4126 in the presence of 10 mM EGTA or 10 mM calcium. Equal amounts of purified Kic1p were run on a gel and Western blotted with anti-Cdc31p antibodies. (C) Coomassie staining of the pure GST–Kic1p fusion proteins as well as copurifying proteins. Purified GST (lane 1, strain MY4124), GST–catalytic domain (lane 2, strain MY4125), and GST full-length fusion proteins (lane 3, MY4126) were separated on an 8% denaturing gel. Lane 4 contains the total yeast extract from MY4126 expressing GST fused to full-length Kic1p. Copurifying peptides of 200, 80, 70, and 40 kD are indicated with asterisks. The GST protein is not observed because it is <30 kD and therefore ran off the gel. Higher percentage gels were run to monitor GST purification (not shown). M, protein molecular weight markers. (D) Anti-GST Western blotting showing full-length GST–Kic1p and several degradation products. Lanes marked 1 and 3 are as in C. (E) Cdc31p binds to full-length GST– Kic1p and its degradation products. Fusion proteins were transferred to membrane and a gel overlay was performed with radiolabeled Cdc31p (Biggins and Rose, 1994). Lanes are marked as in C.

Figure 3

Figure 3

GST–Kic1p kinase activity is Cdc31p-dependent, but calcium-independent. (A) GST–Kic1p and GST alone were expressed in yeast (strains MY4126 and MY4124, respectively) and purified under native conditions by immunoprecipitation with anti-GST antibodies. The kinase activity of GST–Kic1p and GST preparations was assayed at 23°C using γ-labeled ATP. The conditions used are shown above the gel with the addition (+) or omission (−) of the following: the test substrate MBP (myelin basic protein), Mg2+, Ca2+ or EGTA. (B) The kinase activity in the reactions is Kic1p specific. GST, GST-Kic1p, and GST-Kic1-1p were expressed in yeast (strains MY4124, MY4126, and MY4127, respectively) and purified under native conditions by immunoprecipitation with anti-GST antibodies. Kinase assays were performed in standard kinase buffer (see Materials and Methods) using equal amounts of GST–Kic1p or GST–Kic1-1p (proteins amounts were normalized by Coomassie staining and anti-GST Westerns). Protein preparations were assayed for kinase activity at 23°C and 37°C. Both autophosphorylation (auto-P) of GST-Kic1p and phosphorylation of MBP are shown. (C) Cdc31p function is required for GST–Kic1p kinase activity. GST was purified from a CDC31 strain (MY4124) and GST-Kic1p was purified from CDC31, cdc31-1, and CDC31-16 strains (MY4126, MY3899, and MS4048, respectively) under native conditions by immunoprecipitation with α-GST antibodies. Strains containing either cdc31-1 or CDC31-16 are temperature sensitive for growth at 37°C. Equivalent amounts of GST–Kic1p kinase were used in each assay. Phosphorylation of MBP is shown. GST–Kic1p kinase activity is reduced in the presence of mutant Cdc31-1p or Cdc31-16p at 23°C and is at background levels at 37°C. Kinase activities were quantified by PhosphorImager analysis (see Materials and Methods).

Figure 4

Figure 4

kic1 mutants fall into three classes. Wild-type (MS10), kic1-1 (MY3779), kic1-4 (MY4066), kic1-3 (MY4065), kic1-5 (MY4063), kic1-2 (MY4064), and kic1-101 (MY6306) strains were serially diluted and aliquots were spotted onto YPD or YPD containing 1 M sorbitol media (+ sorbitol) and incubated at the indicated temperatures. At 37°C, class A mutants grew poorly compared with the wild type. Class B mutants fail to grow at 37°C but the growth defect was suppressed by the addition of 1 M sorbitol to the media. Class C mutants failed to grow at 37°C in both the presence and absence of 1 M sorbitol.

Figure 5

Figure 5

Images of wild-type and kic1 strains. Wild-type (MS10), kic1-1 (MY3779), kic1-2 (MY4064), and kic1-3 (MY4065) strains were incubated in YPD at 37°C for 8 h, with (+) or without (−) 1 M sorbitol and analyzed microscopically. The asterisks indicate lysed cells or lysed cell contents. Single arrows indicate wide bud necks.

Figure 6

Figure 6

Electron microscopic images of wild-type and kic1 strains. Wild-type (MS10), kic1-1 (MY3779), kic1-2 (MY4064), and kic1-3 (MY4065) strains were grown to exponential phase at 23°C and then shifted to 37°C for 8 h before being fixed and stained for electron microscopy as described in Gammie et al. (1998). Cells in the center and right columns are magnified 3.2× compared with the left column. Chitin rings (CR) and primary septa are indicated (PS). “Frayed” or discontinuous cell walls are indicated with double arrows. Bar in left column, 2 μm.

Figure 7

Figure 7

Morphological defects of cdc31 strains. The cdc31-1 (MY3873), cdc31-2 (MS2260), cdc31-5 (MS2261), and CDC31-16 (MS3510) strains were incubated in YPD 37°C for 8 h and analyzed microscopically. Single asterisks, lysed cells; double asterisks indicate cell debris; double arrows, long narrow bud.

Figure 8

Figure 8

CDC31 and KIC1 interact in vivo. (A) cdc31-1 kic1-1 double mutants have a synthetic lysis defect. The cdc31-1 kic1-1 strain (MY4977) was incubated in YPD at 37°C for 8 h in the presence (+) or absence (−) of 1 M sorbitol and analyzed microscopically. Asterisks, lysed cells. (B) The elongated bud defect of Kic2p overexpression is suppressed by increasing the expression of Cdc31p. Overexpression of the Kic1p (Kic1p O/E) protein induces elongated bud growth (−CDC31 2μ, strain MY4123) that is suppressed when CDC31 is expressed off a high copy vector (+CDC31 2μ, strain MY5493).

Figure 9

Figure 9

kic1 and cdc31 strains have actin localization defects. Wild-type (MS10), kic1-1 (MY3779), kic1-3 (MY4065), kic1-2 (MY4064), cdc31-1(MY3873), and cdc31-1 kic1-1 (MY4977) strains were grown to exponential phase at 23°C, shifted to 37°C for 4 h, and then prepared for immunofluorescence as described in Material and Methods. A representative gallery of cells (unbudded, budded, and large budded) is shown for each strain. Wild-type (WT) cells show a characteristic actin localization pattern during the cell cycle. Unbudded WT cells show no actin concentration. Actin becomes highly concentrated in the emerging small bud of WT cells. Medium-budded WT cells show staining primarily in the bud tip. In WT large-budded cells before cytokinesis, actin redistributes to the septum between the two daughter cells. In the kic1 mutants actin was often prematurely localized to the necks of emerging buds (*) and buds appeared to be wider than normal (**). In addition, kic1 mutants also accumulated actin bars (ab), which are thought to be agglomerations of improperly folded actin. The kic1 mutants also accumulated cells with elongated bud necks containing two constrictions (arrows).

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