Genetic analysis of calmodulin and its targets in saccharomyces cerevisiae.

Key Words calcium, signal transduction, calcineurin, myosin, spindle pole body

* Abstract Calmodulin, a small, ubiquitous [Ca.sup.2+]-binding protein, regulates a wide variety of proteins and processes in all eukaryotes. CMD1, the single gene encoding calmodulin in S. cerevisiae, is essential, and this review discusses studies that identified many of calmodulin's physiological targets and their functions in yeast cells. Calmodulin performs essential roles in mitosis, through its regulation of Nuf1p/Spcll0p, a component of the spindle pole body, and in bud growth, by binding Myo2p, an unconventional class V myosin required for polarized secretion. Surprisingly, mutant calmodulins that fail to bind [Ca.sup.2+] can perform these essential functions. Calmodulin is also required for endocytosis in yeast and participates in [Ca.sup.2+]-dependent, stress-activated signaling pathways through its regulation of a protein phosphatase, calcineurin, and the protein kinases, Cmk1p and Cmk2p. Thus, calmodulin performs important physiological functions in yeast cells in both its [Ca.sup.2+]-bound and [ Ca.sup.2+]-free form.

INTRODUCTION

Calmodulin Structure and Function

Regulated changes in the concentration of cytosolic [Ca.sup.2+] control such diverse biological processes as muscle contraction, fertilization, secretion, cell proliferation, and apoptosis. Calmodulin, a small [Ca.sup.2+]-binding protein, is found in all eukaryotic organisms and is highly conserved. Calmodulin serves as a major intracellular [Ca.sup.2+] receptor and mediates many of the effects of this ion. At resting levels of [Ca.sup.2+], calmodulin exists in the [Ca.sup.2+]-free, or apo-calmodulin form. In response to a [Ca.sup.2+] signal, calmodulin binds [Ca.sup.2+] and consequently undergoes a conformational change that allows it to bind to and activate a host of target enzymes. Since its discovery in 1970 (14), the mechanism of [Ca.sup.2+]/calmodulin-dependent regulation of target enzymes has been characterized extensively through in vitro biochemical and structural analyses. More recently, studies of calmodulin in several genetically tractable organisms established that calmodulin is required for viab ility (25, 123, 142). Genetic dissection of calmodulin function in the yeast Saccharomyces cerevisiae has added significantly to our understanding of this regulator by identifying physiologically relevant targets of calmodulin (Table 1), and by establishing the functional significance of both [Ca.sup.2+]-bound and [Ca.sup.2+]-free calmodulin in vivo.

Calmodulin contains four copies of a [Ca.sup.2+]-binding motif known as an EF-hand, each of which binds one [Ca.sup.2+] ion. An EF-hand is made up of a 12-residue [Ca.sup.2+] binding loop flanked by two [alpha]-helices (60). Within the loop, [Ca.sup.2+] is coordinated by oxygens on six different amino acid residues. Calmodulin is one member of a large class of EF-hand-containing [Ca.sup.2+]-binding proteins (98). S. cerevisiae contains five calmodulin-related proteins, each of which contains four EF-hand motifs: Cdc3lp, is a component of the yeast microtubule organizing center and the yeast homologue of centractin (5). Mlclp and Mlc2p are myosin light chains, which regulate distinct yeast myosins (6, 136). Cnblp encodes the regulatory subunit of the [Ca.sup.2+]/calmodulin-regulated phosphatase, calcineurin (22,62). Frq1p encodes the regulatory subunit of a phosphatidylinositol-4-OH kinase and is a homologue of frequenin, a protein found in vertebrate neurons (46).

EF-hand-containing proteins typically undergo a structural change upon binding [Ca.sup.2+]; however, this conformational change differs substantially for each class of EF-hand protein (155). Structural analyses have established that calmodulin is a dumbbell-shaped molecule with two similar domains, each containing two EF-hand [Ca.sup.2+]-binding motifs, connected by a short flexible linker. In the absence of [Ca.sup.2+], the EF-hands are in a "closed" conformation. This [Ca.sup.2+]-free form of calmodulin is able to bind to a subset of target proteins. [Ca.sup.2+] binding causes a change to an "open" conformation, which also results in exposure of two hydrophobic surfaces that allow calmodulin to bind to its [Ca.sup.2+]-dependent target proteins (reviewed in 155). Binding sites for calmodulin share limited sequence homology, but are similar in structure, and are typically regions 20 amino acids long that form basic amphipathic [alpha]-helices. Several types of calmodulin-binding sites can be distinguished bas ed on the spacing of particular bulky residues and the tendency to bind apo-calmodulin (18, 125).

Identification and Characterization of Calmodulin from S. cerevisiae

Calmodulin was purified from S. cerevisiae based on the similarity of its physical properties to those of vertebrate calmodulin (25, 72, 107). Once purified, partial amino acid sequence was determined and used to synthesize oligonucleotide probes to identify the gene, CMD1,from a yeast genomic library (25). S. cerevisiae contains a single calmodulin gene that is required for viability and encodes a protein 60% identical to vertebrate calmodulins (25). The primary structure of yeast calmodulin is like its vertebrate counterpart, having four predicted helix-loop-helix EF-hand domains distributed similarly in the protein sequence. However, there are significant differences in the structure and [Ca.sup.2+]-binding properties of yeast and vertebrate calmodulins. Vertebrate calmodulin binds four molecules of [Ca.sup.2+] per molecule of calmodulin, whereas yeast calmodulin binds a maximum of three molecules of [Ca.sup.2+] (72, 76, 133). The most C-terminal EF-hand in yeast calmodulin (site IV) has a deletion of one residue in the [Ca.sup.2+]-binding loop and also contains a substitution of glutamine for a highly conserved glutamate at position 12. Thus, while this region of the protein still maintains the helix-loop-helix conformation found in other calmodulins, site IV is defective for [Ca.sup.2+] binding (77, 133). The other EF-hands in yeast calmodulin bind [Ca.sup.2+] with high affinity ([K.sub.d] = 2-5 x [10.sup.-6] M), although the exchange rate of [Ca.sup.2+] for these sites is slower than that observed for vertebrate calmodulins (133). In its [Ca.sup.2+]-bound form, yeast calmodulin exists in a more compact form than do its vertebrate counterparts (157), owing to interactions between the N-terminal and C-terminal domains (63). Despite these differences in biochemical properties, vertebrate calmodulin is able to complement the essential function of calmodulin in yeast (24, 45, 103).

GENETIC ANALYSIS OF CALMODULIN FUNCTION

Analysis of Conditional Mutants

The identification and characterization of calmodulin in S. cerevisiae made possible an extensive genetic dissection of calmodulin function. Examination of conditional calmodulin mutants identified multiple distinct essential functions for this protein in vivo. Initially, two temperature-sensitive calmodulin mutants were studied: cmdl-1, which contains two amino acid substitutions (23) and cmdl-101, which contains an allele engineered in vitro to allow overexpression of a truncated calmodulin lacking its N-terminal half in the yeast genome (139). Temperature-shift experiments with both strains revealed a requirement for calmodulin primarily during nuclear division; at the nonpermissive temperature, cells with a duplicated DNA content accumulated, and further analysis of these cells identified abnormalities in spindle morphology (23, 139). A defect in bud growth was also observed for cmd1-1. The consequences of depleting calmodulin in vivo, i.e., shutting off expression of CMD1 driven by a galactose-regulated promoter, were similar to the effects observed in the temperature-sensitive calmodulin mutant strains (104). Thus, the first essential role demonstrated for calmodulin was in nuclear division.

Further genetic analysis by Ohya & Botstein established that calmodulin has several additional essential functions. A panel of conditional mutations was generated by changing conserved phenylalanine residues of calmodulin to alanine (105). The resulting collection of mutants fall into four distinct phenotypic groups, cmd1A-D, that also display intragenic complementation. Members of one of the groups (cmd1C) exhibit a defect in mitosis similar to that described for cmd1-1 and cmd1-101. Other mutants reveal an essential role for calmodulin in bud emergence (cmd1D) and actin localization (cmd1A) (105). In the final group of mutants (cmd1B), the characteristic pattern of calmodulin localization is disrupted (105). Calmodulin localizes to sites of bud formation, bud tips, and the bud neck in vivo (10,138). This cellular distribution overlaps in part with that of actin patches and reflects calmodulin's role in polarized growth. In cmd1B mutants, however, calmodulin is distributed diffusely throughout the cell.

The distinct nature of the four phenotypic groups of cmd1 mutants and their ability to complement each other suggested that each group was compromised for activation of different essential target(s) in vivo. Further analyses have confirmed that cmd1C mutants are specifically compromised for regulation of Nuf1p. However, other mutant groups may be deficient for activation of more than one target (see section on [Ca.sup.2+]-independent Targets). Nonetheless, this panel of mutants has been a powerful tool for analyzing and characterizing the diverse functions of calmodulin in vivo.

Analysis of [Ca.sup.2+]-Binding--Defective Mutants

The essential role of calmodulin in vivo was expected to depend on its ability to bind [Ca.sup.2+], because the role of this protein as an intracellular [Ca.sup.2+] sensor was so well established. However, this notion was challenged by the finding that mutant alleles of CMD1 that are completely defective for [Ca.sup.2+]-binding support yeast growth (39). [Ca.sup.2+]-binding-defective alleles were constructed by directed substitution of amino acid residues required for [Ca.sup.2+] ion coordination, and in vitro analyses confirmed that the resulting proteins (cmd1-3p, cmd1-6p) were deficient for [Ca.sup.2+] binding. Surprisingly, yeast cells whose only source of calmodulin are these [Ca.sup.2+] binding-defective-mutant proteins show minimal disruptions in growth and morphology under standard culture conditions (39). The intracellular localization of the mutant proteins is also indistinguishable from that of wild-type calmodulin (10, 92). Although [Ca.sup.2+] independent binding of mammalian calmodulin to severa l proteins had been demonstrated previously, the physiological relevance of these interactions was not fully appreciated. The studies in S. cerevisiae clearly established that for this organism the essential functions of calmodulin do not depend on its ability to bind [Ca.sup.2+], and later identification of the essential targets, Nuf1p/Spc110p and Myo2p, have confirmed their [Ca.sup.2+]-independent interaction with calmodulin (see section on Targets of Calmodulin).

Unfortunately, these findings are often misinterpreted as indicating that there are no [Ca.sup.2+]-independent functions for calmodulin in S. cerevisiae and that this yeast is devoid of [Ca.sup.2+]-dependent signaling pathways. However, as discussed below, the activation by calmodulin of at least two different target proteins, calcineurin, the [Ca.sup.2+]/calmodulin-dependent phosphatase, and calmodulin-regulated kinases (Cmk1p and Cmk2p), is [Ca.sup.2+] dependent, and the [Ca.sup.2+]-binding--defective calmodulin mutants fail to activate these targets in vivo (20, 93). However, under standard laboratory conditions, neither the [Ca.sup.2+]/calmodulin-dependent phosphatase nor the kinase is required for viability (21, 69, 106, 111).

TARGETS OF CALMODULIN

[Ca.sup.2+]-Independent Targets

Nuf1p/Spcll0p Calmodulin localizes to the spindle pole body (SPB), the yeast microtubule organizing center (MTOC), throughout the cell cycle, and the essential target of calmodulin in mitosis, Nuf1/Spc110p, is a component of the SPB (38, 92, 137). The SPB is embedded in the yeast nuclear envelope and is the sole organelle responsible for nucleating nuclear and cytoplasmic microtubules. Thus, the SPB is equivalent in function to the centrosome of animal cells and there is substantial similarity among the protein components of these two organelles…