The Correlation Between Levels of cAMP and Cell Lysis

Radioimmunoassay has revealed that the srbl-1 mutation leads to an increase in cellular cAMP. It has also been demonstrated that mutations in PDE2 and RAS2,

which are both components of the iM5'-cAMP pathway, give rise to osmotic shock sensitivity. These results imply that the phenotypes of these mutations are, in part, mediated through the changes they effect on cellular cAMP concentration.

3.5.1 Direct Comparison of cAMP Assay and Lysis Data

A bar graph was drawn in which the cAMP level for each strain investigated was plotted alongside the extent of cell lysis (see figure 6).The two wild type strains ,

1 2- 1 0- •a_lU O 4 2- S288C - 0 .8 s ) -0.6 3 -0.4 sr

4STIA 4SLUP YLGl J105 DJ12-9D YLG3TK161-R2V Y e a s t S t r a i n s

Fig. 6. Correlation between levels of intracellular cAMP and lysis ability. The left hand y axis refers to the solid blocks and the right hand y axis to the shaded blocks.

of lysis upon osmotic shock. The same is also true for the pdel mutant. On the other hand , pde2 mutants constructed both for this study and others (7d and DJ12-9D respectively) have higher levels of cAMP and significant lysis ability following osmotic shock. The disruption of both PDEl and PDE2 in (strain YLG3) results in an increase in cAMP level and higher, but not proportionately higher, lysis yields compared to the corresponding single mutants in the same genetic background. The RAS2^^^^^ strain TK161-R2V had the expected increased levels of cAMP and also has a demonstrable osmotic shock sensitivity when compared to the isogenic wild type strain, YLGl. However, the lysis ability of the RAS2^‘^‘^^ mutant is significantly lower than that of the srbl-1 and

pde2 single mutants. It can clearly be seen that increasing levels of cAMP lead to an increase in osmotic instability. However, the condition cannot be explained simply in terms of gross intracellular cAMP concentrations. The srbl-1, pde2

double mutant has almost the same levels of cAMP as the two single mutants, yet it shows a substantially increased lysis ability to that of either mutation alone.

3.5.2 Exogenous cAMP Promotes Cell Lysis

Direct evidence for the involvement of cAMP in the maintenance of the cellular integrity would be provided if it could be demonstrated that the addition of the cyclic nucleotide to yeast cells increased their susceptibility to lysis upon osmotic shock. There is some controversy as to whether or not Saccharomyces

cerevisiae is permeable to cAMP. It is generally acknowledged that yeast is

impermeable to this compound, although contradictory data has been submitted (Singh et al, 1980) and evidence has been presented that cAMP uptake may be

induced under certain physiological conditions, especially growth at low pH (Mahler and Lin, 1978). However, the cAMP-utilising mutants caml, 2 and 3

(Matsumoto et ah, 1982) have provided the only pertinent data about the effect of exogenous cAMP on yeast cell metabolism. Interestingly, the only other class of mutants which have been clearly demonstrated to respond to exogenous cAMP are the pde2/rcal mutants (Wilson and Tatchell, 1988; Wilson et al,

1993; Mitsuzawa, 1993).Wilson et al (1993) observed that the presence of exogenous cAMP raises the intracellular cAMP levels in pde2 strains and have suggested that the role of the high-affinity phosphodiesterase may be to degrade extracellular cAMP in order to maintain a threshold for cell-to-cell signaling mediated by the cyclic nucleotide. In order to see if exogenous cAMP would have an effect on the lysis phenotype, the strains were grown in the presence of 4 mM cAMP. Equal volumes of YEPD 10% sorbitol, one containing 4 mM cAMP, were inoculated with exponential phase cells to an ODeoo 0.1. The cultures were incubated at 30°C until an ODôoo of approximately 0.55 was reached at which point the cells were harvested and the lysis test was carried out (2.5.7). The results are shown in Table 10.

From the Table, it can clearly be seen that exogenous cAMP causes an increase in cell lysis, even with wild type cells. The results however are not conclusive with the magnitude of the effect (15-20%) not being large enough to be considered significant. It is not known by what extent the intracellular levels of cAMP were raised by the addition of exogenous cAMP, as this was not

OD260 Released

Strain No cAMP 4 mM. cAMP % increase

S288C 0.256 0.306 17.7

7d 1.75 2.25 29.4

DJ12-9D 5.16 5.88 14.0

4STLU 4.42 5.69 28.7

4SLUP 5.23 6.175 18.1

measured. Likewise, cAMP uptake from the medium was not measured (in both cases this was due to financial constrainst due to the expense of the cAMP assay system). Therefore it cannot be concluded that this increase in lysis is mediated through an increase in cellular cAMP. A further examination of a range of cAMP concentrations may have proved rewarding, but it should also be noted that as a media constituent, cAMP would be very expensive, especially on a larger scale. Other ways of increasing the intracellular levels of cAMP such as glucose induction (van der Plaatl974) and temperature shift (Boutlet et al,

1985, Camonis et al, 1986 and Matsumoto et al, 1985) were considered, but not investigated since no supporting cAMP data could have been provided.