FEMS Microbiology Letters 41 (1987) 253-255 253 Published by Elsevier

FEM 02739

Calmodulin levels in yeasts and filamentous fungi

G a n a p a t h y M u t h u k u m a r *, A n n W. Nickerson , and K e n n e t h W. N icke r son

School of Biological Sciences, University of Nebraska, Lincoln, NE, U.S.A.

Received 12 December 1986 Accepted 22 December 1986

Key words: Ceratocystis ulmi; Calmodulin; Yeast; Filamentous fungus; Fungal dimorphism

1. SUMMARY

A calmodulin-specific radioimmunoassay was used to measure the calmodulin present in 5 fila- mentous fungi and 14 yeasts. The filamentous organisms contained from 2.0 -6.5 gg calmodulin per mg soluble protein, whereas the yeasts con- tained from 0.02-0.89 gg per mg. The size of this difference suggests that the evolutionary transition to yeasts included loss of the extra calmodulin synthetic capacity necessary for mycelial growth.

In C. ulmi, a Ca(II)-calmodulin interaction was necessary for mycelial growth and the absence of this interaction led to growth in the yeast phase [1]. Because of its role in the regulation of dimor- phism, we thought that calmodulin might be re- lated to the evolutionary distinction between the filamentous fungi and budding yeasts. The present paper reports the calmodulin levels present in 14 yeasts and 5 filamentous fungi as determined by a calmodulin-specific radioimmunoassay.

2. IN T R ODUC TI ON

Fungi include many attractive systems for studying the molecular level parameters regulating cell shape. Comparisons can be made among bud- ding yeasts, filamentous fungi, and dimorphic fungi. We have previously shown [1-3] that the Ca(II)-calmodulin complex functions as an o n / o f f switch regulating yeast-mycelial dimorphism in the ascomycete Ceratocystis ulmi (Buism.) C. Moreau, the causative agent of Dutch elm disease.

Correspondence to: Kenneth W. Nickerson, School of Biologi- cal Sciences, University of Nebraska, Lincoln, NE 68588-0118, U.S.A. * Present address: Department of Microbiology and Public

Health, Michigan State University, East Lansing, MI 48824, U.S.A.

3. MATERIALS AND METHODS

The origins of the fungi employed in the calmodulin determinations have been described previously [4,5]. Cultures were inoculated from malt-yeast extract-dextrose agar slants into 50-ml flasks containing 10 ml of malt extract broth (Difco, Detroit) and grown at 25 °C for 2 days. These starter cultures (1 ml) were used to inoc- ulate 250-ml flasks containing 50 ml of malt ex- tract broth or Czapek Dox broth (Difco, Detroit). The cultures were grown with rotary agitation at 25°C on a New Brunswick Scientific Co. G52 shaker at 220 rpm. In each case, dense growth was achieved after 2 days and the cells were harvested after 3 days. These stationary phase cells were examined microscopically; no pseudomycelia were observed.

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Calmodulin levels were determined with a ca lmodul in-spec i f ic r a d i o i m m u n o a s s a y kit (Amersham, Arlington Heights, IL) as described previously [2]. Cells were harvested 3 days after inoculation, washed 3 times in sodium phosphate buffer (pH 6.5) and resuspended in the Amersham calmodulin-extraction buffer supplemented with 5 mM EDTA and 0.6 mM phenylmethylsulfonyl fluoride (PMSF). The cells were disrupted for 1 min at 4000 rev. /min in a Braun MSK homo- genizer with 0.3-mm-diameter glass beads. Cell breakage was at least 90% complete. All subse- quent steps were as described in the RIA kit, with the calmodulin levels expressed as micrograms per milligram of soluble protein. Total protein in the supernatant was determined by the method of Bradford [6].

Table 1

Calmodulin levels in yeasts and filamentous fungi

Organism Calmodulin (~ g / m g protein) a

Filamentous fungi Aspergillus niger 3.5 tlelminthosporium sp. 6.5 Penicillium notatum 2.0 Rhizol)us arrhizus 3.3 Trichoderma lignorum 2.0

Yeasts Candida parapsiolosis 0.60 Citeromyces matritensis 0.39 Debar),omyces vanriji 0.29 Itansenula anomala 0.15 ttansenula subpelliculosa 0.12 Kluyveromvces wickerhamii 0. l0 Nadsonia fuh~escens 0.89 Pact~vsolen tannophilus 0.45 Rhodotorula sp. 0.60 Saccharomyces cereoisiae b 0.03 Saccharomyces eerevisiae 0.08 Saccharomw'es cerevisiae 0.09 Saccharomvces cerevisiae 0.11 Schizosaccharornyces octosporus 0.02 Schwanniomyces alluvius 0.57 Tremella mesenterica 0.45 Trigonopsis variabilis 0.50

a Values reported are the average of duplicate experiments agreeing within ± 10%.

b Three independent cultures of S. cerevisiae were tested. The fourth value is for commercial Red Star Bakers Yeast.

4. RESULTS

Table 1 shows the calmodulin levels detected in 5 filamentous fungi and 14 yeasts. Calmodulin was found in each of the fungi tested. The fila- mentous organisms contained from 2.0-6.5 /~g calmodulin per mg soluble protein whereas the yeasts contained from 0.02-0.89 t~g per mg. These results were obtained with cells grown on malt extract broth. Six cultures Rhizopus arrhizus, Candida parapsiolosis, Debaryomyces vanriji, Kluyveromyces wickerharnii, Rhodotorula sp., and Saccharomyces cereoisiae, were also able to grow on the defined Czapek-Dox broth. The calmodulin levels detected in these cells were similar to those found in the cells grown in malt extract. The use of a defined medium combined with extensive washing was important because the malt extract medium itself gave low calmodulin values (0.02 /~g/mg protein) with the radioimmunoassay method.

5. DISCUSSION

Fungal calmodulins have previously been de- tected in Achlya ambisexualis [7], Agaricus bi- sporus [8], Agaricus campestris [9], Blastocladiella emersonii [10], C. ulmi [1,2,11], Coprinus lagopus [9], Cortinarius sp. [8], Dictyostelium discoideum [12], Mucor rouxii [13], Neurospora crassa [14], Phycomyces blakesleeanus [15], Physarum poly- cephalum [16], and Russula sp. [8]. Our data have extended this list to include 5 more filamentous fungi and 14 yeasts. The presence of calmodulin in every fungal species examined reinforces the gen- eralization that calmodulin is present in all eukaryotic organisms. In particular, their presence in 14 yeasts confirms reports of calmodulin in S. cerevisiae [9,17] and the yeast form of Candida albicans [17]. Together, these results invalidate the simplistic model that yeasts are yeasts because they lack calmodulin.

Our data are the first to compare cellular calmodulin levels in the fungi by a single uniform criterion. They reveal dramatic differences be- tween the levels of calmodulin present in yeasts and filamentous fungi. These differences are most

likely real. Firstly, our calmodulin extraction buffers were supplemented with the protease in- hibitors EDTA and PMSF. Secondly, calmodulin is such a highly conserved protein that the radio- immunoassay is known to be immunologically cross-reactive for calmodulins from vertebrate, in- vertebrate, and plant sources [18], as well as from fungi [2]. The S. cerevisiae calmodulin sequence is 60% identical and 80% homologous with that of bovine brain calmodulin [19]. Thirdly, the radio- immunoassay employed detects both active and inactive calmodulins. With the dimorphic fungus C. ulmi, the total presence of calmodulin de- termined by radioimmunoassay [2] closely ap- proximated the biological activity of calmodulin determined by stimulation of calmodulin-deficient cAMP phosphodiesterase [11]. We found up to 14.2 /~g calmodulin per mg soluble protein by radioimmunoassay ]2] and about 17.1 /~g per mg by enzyme activation [11]. These comparatively high calmodulin concentrations suggest that calmodulin fulfils a sustained structural role in mycelial development, not just a transitory en- zyme activation.

The magnitude of the yeast vs. filamentous differences (Table 1) is consistent with the view that the relatively few yeast species evolved from the more numerous filamentous fungi and that decreased calmodulin levels were a necessary part of the filamentous to yeast transition. Our data pertain to calmodulin protein levels, not gene copy number. However, our yeast versus filamentous differences in the calmodulin protein levels prob- ably reflect underlying gene differences as well for two reasons. Firstly, S. cerevisiae contains a unique single-copy calmodulin gene residing on chro- mosome II [19]. Disruption of the calmodulin gene with the URA3 gene resulted in a recessive lethal mutation [19], indicating that calmodulin is re- quired for the growth of yeast cells. Secondly, at least in S. cerevisiae, the calmodulin gene does not appear to exhibit dosage compensation--in- creased copy number gives increased protein activ- ity [20].

We have been unable to discern any physiologi- cal or taxonomic themes distinguishing those yeasts containing very low calmodulin levels (~<

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0.15 /~g/mg protein) from those containing com- paratively high levels (>/0.45 /~g/mg protein). Possibly the cell cycle arrest point in stationary phase may influence the levels of calmodulin de- tected. In both mammalian cells [21] and C. ulmi [2], calmodulin synthesis occurred at the G1-S boundary of the cell cycle.

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