FEMS Microbiology Letters 60 (1989) 283-288 283 Published by Elsevier

FEM 03644

Stress-induced protein synthesis in Chlamydomonas reinhardtii

Paul Nicho l son and Chr i s tophe r J. H o w e

Department of Biochemistry, Unioersity of Cambridge, Cambridge, U.K.

Received 21 March 1989 Accepted 3 April 1989

Key words: DNA damage; 4-Nitroquinoline-N-oxide; Heat shock; Protein synthesis; Chlamydomonas reinhardtii

1. SUMMARY

Following exposure to UV light the synthesis of six polypeptides (112, 100, 89, 76, 71 and 65 kDa) was found to be enhanced or induced in the alga Chlamydomonas reinhardtii. Treatment with 4- nitroquinoline-N-oxide (NQO) resulted in the en- hanced/ induced synthesis of six polypeptides with molecular masses similar to those enhanced fol- lowing exposure to UV light. Heat shock resulted in the enhanced synthesis of five polypeptides (89, 76, 71, 60 and 22 kDa), three of which (89, 76 and 71 kDa) had apparently identical mobilities to polypeptides whose synthesis was enhanced fol- lowing UV treatment.

2. IN TR ODUC TI ON

Prokaryotic and eukaryotic cells possess induci- ble responses to environmental stresses. Two of the best studied of these are the SOS response and the heat shock response, both of which involve the rapid and generally transient synthesis of specific

Correspondence to: Dr. C.J. Howe, Department of Biochem- istry, University of Cambridge, Tennis Court Road, Cam- bridge CB2 1QW, U.K.

sets of proteins. The SOS response has been best characterised in Escherichia coli, where it involves the induction in response to DNA damage of at least 17 genes, including recA, and leads to in- creased levels of DNA repair and recombination [1]. The heat shock response has been observed in all species examined to date, and there is consider- able homology between the proteins produced in very divergent organisms [2,3]. Results from organisms such as E. coil, Drosophila melanogaster and Saccharomyces cereoisiae indicate that the responses to heat shock and DNA-damaging agents overlap in that certain genes respond to more than one stress stimulus [4,5,6]. For example, in E. coli two such genes are groEL and dnaK (gene products 61 kDa and 73 kDa respectively). It is thought that the dnaK protein may be in- volved in switching off the heat shock response, possibly through protease activity on the sigma-32 subunit of RNA polymerase [7,8]. This and the groEL gene product appear to be involved in the assembly a n d / o r disassembly of protein-contain- ing complexes [9,10].

As part of a study of DNA repair and recombi- nation in algae and higher plants we have at- tempted to identify proteins in Chlamydomonas, whose synthesis is enhanced in response to DNA damage. The DNA-damaging agents used here are

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UV light and 4-nitroquinoline-N-oxide (NQO) which produces UV-mimetic lesions in DNA by the formation of guanine adducts [11]. Use of thc latter may indicate which polypeptides are in- duced as a result of D N A damage rather than any of the other effects that might be postulated for UV light, such as damage to the photosynthetic apparatus. We demonstrate here the enhancement of synthesis of several polypeptides in response to DNA-damaging treatments. However, heat shock also leads to the enhancement of synthesis of specific proteins in algae and higher plants [12,13,14], and wc show here that three of the polypeptides whose synthesis is enhanced after DNA damage may be similarly affected by heat shock. They may therefore be less directly associ- ated with DNA repair, although there is some evidence that heat shock causes DNA strand breakage in mammalian cells [15].

3. MATERIALS AND METHODS

Chlamydomonas reinhardtii wild-type strain Paris was grown axenically on the (Tris-acetate- phosphate) medium of Gorman and Levine [16]. Cultures were maintained at 25°C in an orbital incubator and illuminated with cool white light at an intensity of 200 Lux. Mid-log phase cells (2 × 106 cells/ml) were sedimented by centrifugation for 5 min at 12000 x g and resuspended in half the volume of supernatant for use in experiments.

Cells were exposed to doses of UV light of 130--400 J / m 2 from a germicidal lamp. The effect of UV dosage on cell viability was evaluated for mid-log phase cells by observing fluorescein di- acetate fluorescence of cells 6 h after treatment. Aliquots (1 ml) or irradiated or unirradiated cells were returned to the incubator for 1 h before the addition of 150 t.tCi [35S]-methionine (Amersham International; 15 mCi/ml , 1200 Ci/mmol) . The labelled cells were incubated for a further 2 h before harvesting by centrifugation. The pellet was resuspended in 100 /~1 of electrophoresis sample buffer (8% sodium dodecyl sulphate, 20% glycerol, 0.01% bromophenol blue, 0.2 M Tris-HC1 pH 6.8) containing 1 mM phenylmethylsulphonyl fluoride as a protease inhibitor and 10 ~1 of ]~-mercapto-

ethanol. Cells were then lysed by sonication for 30 sec in a sonicating bath.

NQO treatment was carried out by incubating cells in the presence of NQO at concentrations from 0.5- 5.0/~g/ml for 1 h prior to labelling and harvesting as for UV-irradiated cells. Cells were heat-shocked by incubation at 38°C for 1 h be- fore addition of [35S]-methionine and further in- cubation for 2 h at the elevated temperature and harvesting as for the other treatments.

Protein extracts were solubilised at 37 °C for 30 min and cell debris removed by centrifugation for 5 min at 20000 x g prior to electrophoresis. The protein supernatants were analysed by electro- phoresis through 10-20% linear gradient SDS- polyacrylamide gels [171 and labelled polypeptides detected by fluorography [18]. Dried gels were placed against pre-flashed X-ray film (Kodak X- Omat S) and exposed at - -70°C for 3-6 days before developing. The relative molecular masses of polypeptides were determined by comparison with ]4C-labelled proteins (Amersham Interna- tional) after co-electrophoresis.

4. RESULTS AND DISCUSSION

A kill-curve was produced for C. reinhardtii following exposure to UV light (Fig. 1). In stress experiments a dosage was used that allowed a high

100 •

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75

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25,

I 2 3 4 5

U V D o s e ( k J / m 2 )

Fig. l. Graph showing the effect of UV dose from a germicidal lamp (245 nm) upon survival of cells of C. reinhardtii.

285

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-71

-65 .112 .100 "89 "76 -71 "65

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Fig. 2. Effects of UV irradiation on protein synthesis. Tracks in 2a show the polypeptide synthesis profile of (1) unirradiated cells labelled with [3sSl-methionine for 2 h; (2) cells irradiated with 270 J/m 2 UV light and labelled for 2 h or (3) incubated for 1 h prior to labelling. Tracks in 2b afford resolution of high molecular weight polypeptides and show profiles from (1) unirradiated cells and (2) cells irradiated with 130 J/m 2 UV light. Enhanced/induced polypeptides are indicated. Positions of molecular weight standards (kDa) are given on the left of

the tracks.

- - - , " - .n

% :,..-%'-t ~- , ~ • . ; ,

.. _2..' ~

level ( > 90%) of survival. Such levels of irradia- tion have been successfully employed with yeast to detect the induct ion of gene expression following UV-induced D N A damage [19,20]. In the present study, increases in the level of UV irradiation were not observed to alter the polypept ide profile fur- ther, but did cause a large reduct ion in the incor- pora t ion of radio-label (data not shown). Protein synthesis was significantly reduced in cells im- mediately following exposure even to very low doses of UV light (Fig. 2a), and for this reason a 1-h incubat ion period was included between treat- ment and the addit ion of [35S]-methionine. This pre- incubat ion did not affect the polypept ide pro- file observed.

In prel iminary experiments the synthesis of two polypeptides, 71 kDa and 65 kDa, was found to

be enhanced following irradiation (Fig. 2a) [21]. Subsequently, better resolution of larger poly- peptides was achieved, and synthesis of several additional polypeptides (76, 89, 100 and 112 kDa) was found to be enhanced (Fig. 2b). The synthesis of certain polypeptides was specifically reduced by UV light.

The concentrat ions of N Q O used were similar to those used in experiments with yeast [22]. Higher concentrat ions of N Q O are required to affect visi- bly the polypept ide synthesis profile of suspension cultures of higher plants (J.W. Forster, personal communicat ion) , but with Chlamydomonas rein- hardtii further increases in N Q O concentra t ion resulted in negligible incorporat ion of label into polypeptides and no further change in the profile of synthesis. One-dimensional SDS-polyacryla- mide gel electrophoresis of protein extracts f rom N Q O - and UV-treated cells exhibited identical polypeptide synthesis profiles (Fig. 3), suggesting

9 2 -

6 9 -

4 6 -

3 0 -

1 2 3 4

-112 - 100 - 8 9 - 7 6 -71

- 6 5

Fig. 3. Effects of NQO treatment on protein synthesis. Tracks show polypeptide synthesis profiles of (1) unirradiated cells; cells incubated for 3 h in the presence of (2) 0.5 ~g/ml or (3) 2 ~g/ml NQO; and (4) cells irradiated with 130 J/m 2 UV light and incubated for 1 h prior to labelling. Enhanced/induced polypeptides are indicated. Positions of molecular weight

standards (kDa) are given on the left of the figure.

286

-89 -76 -71

-60

-22

2 3

- 8 9

-76 -71

a b

Fig. 4. Comparison of effects of heat shock and UV irradiation on protein synthesis. Tracks in 4a show polypeptide synthesis profile of (1) control cells of C reinhardtii and (2) cells given a heat shock of 38°C for 3 h. Tracks in 4b show the polypeptide synthesis profile of (1) control cells; (2) cells incubated at 38°C for 3 h; (3) cells irradiated with 130 J / m 2 UV light and incubated for 1 h prior to labelling. Enhanced/ induced poly- peptides are indicated. Positions of molecular weight standards

(kDa) are given on the left of the tracks.

that these two treatments induce a similar re- sponse in C. reinhardtii.

Heat shock at 38°C for up to 3 h also resulted in an alteration of the polypeptide synthesis pro- file, with major induced polypeptides of 89, 76, 71, 60 and 22 kDa (Fig. 4a). This is similar to that observed by other workers [13]. No polypeptides of higher molecular weight were seen on heat shock, although the gel system would have been capable of resolving them. When samples were run in adjacent gel lanes three of the polypeptides (89, 76 and 71 kDa) had apparently identical mobili- ties to polypeptides induced on UV treatment (Fig. 4b). Attempts to confirm the similarity using the 2-D electrophoresis system of O'Farrell [23] were unsuccessful. The polypeptides could not be seen in such gels, which may indicate that both

sets are very basic and failed to enter the first dimension isoelectric focusing gel.

Both UV irradiation and treatment with NQO lead to enhanced synthesis of 112, 100, 89, 76, 71 and 65 kDa polypeptides. Polypeptides of 89, 76 and 71 kDa (which may be related or identical to the UV- and NQO-induced ones) are also induced in response to heat shock, along with others of 60 and 22 kDa (the latter has been implicated in protection against photoinhibition [12]). The 76 or 71 kDa polypeptides may be related to the 73 kDa dnaK gene product of E. coli, which is also in- duced in response to both DNA damage and heat shock. This might be tested immunochemically. We suggest that the 112, 110 and 65 kDa poly- peptides may be more directly associated with DNA damage, and may function in repair a n d / o r recombination processes.

A C K N O W L E D G E M E N T S

This work was supported by the A.F.R.C. We are grateful to Dr. J.W. Forster for helpful discus- sions and advice.

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