The impact of Ras1 C-terminal modification and localisation on signalling in fission yeast
5.2. QUANTIFYING RAS1 LOCALISATION
and OE images
pREP3x-GFP-Ras1 JY1390 (gfp-ras1)
Figure 5.1. Comparing the localisation of GFP-Ras1 when expressed from he ras1 locus and pREP3x
Images of GFP-Ras1 expressed from theras1 locus were taken using a Nikon E800 epifluorescence microscope fitted with an Andor EM-CCD camera, as GFP-Ras1 was not detected using a scanning confocal microscope (Leica SP5). Images of GFP-Ras1 expressed from pREP3x were collected using a Leica SP5 scanning confocal microscope. The scale bar represents 10 μm.
of ImageJ is to analyse the distribution of fluorescence in a cell, with a specific focus on fluorescence at the cortex of the cell. This analysis therefore allows
the quantification of plasma membrane localisation. Images of Δras1 cells
(JY1279) expressing GFP and GFP-Ras1 from pREP3x were obtained using a Leica SP5 scanning confocal microscope (Figure 5.2A). Quimp software was then used to separate fluorescence at the periphery of the cell from the interior, and the intensity of fluorescence in each of these compartments was determined (Figure 5.2B). The level of plasma membrane fluorescence in cells expressing
GFP alone was determined to be 3.1 + 1.0 %, representing a small proportion
of soluble GFP in close proximity to the membrane. By contrast, GFP-Ras1
displayed predominantly plasma membrane localisation (78.0 + 3.5 %) (Fig-
ure 5.2C). Examination of GFP-Ras1 localisation indicates that the majority of GFP-Ras1 not at the plasma membrane was located on endomembrane structures. These observations are in agreement with those made in previ- ous studies, in which it was reported that Ras1 localised mainly to the plasma
Ras1-GFP pGFP pGFP-Ras1 pGFP pGFP-Ras1 *** A B C
Figure 5.2. Quantifying the localisation of GFP-Ras1 using Quimp software
Images ofΔras1 cells (JY1279) expressing GFP and GFP-Ras1 from pREP3x were obtained using a Leica SP5 scanning confocal microscope (A). The intensity of fluorescence at the periphery compared to the interior of the cell was determined using Quimp software (B), and percentage plasma membrane fluorescence was calculated (C). The analysis indicated that GFP-Ras1 displayed predominantly plasma membrane fluorescence. Statistical significance was determined using a one-way anova with a Tukey multiple comparison post test. Three asterisks indicates a P-value of 0.001. The scale bar represents 10μm.
A B Vector p p Vector p p B 60 70 80 90 100 110 120 130 140 JY544 JY1247 Integrated Ras1S22N Integrated Ras1Q66L Integrated Ras1C215S Q66L -9 -8 -7 -6 -5 -4 Integrated Ras1G17V Log [P-Factor] M
Figure 5.3. Pheromone-dependent changes in transcription and cell volume in cells expressing GFP-Ras1 from pREP3x
Pheromone-responsive changes in transcription and cell volume were observedΔras1 (JY1279) cells transformed with pREP3x-Ras1, pREP3x-GFP-Ras1 and vector alone. Cells were grown in the presence of 1 nM to 100 μM pheromone in DMM for 16 h. Assays were then performed for β-galactosidase activity (A) and average cell volume (B). Both Ras1 and GFP-Ras1 displayed comparableβ-galactosidase ac- tivity, and both displayed an increase in cell voume upon pheromone stimulation. Data shown is an average of three independent determinants (+SEM).
5.2.3. Expression of GFP-Ras1 from pREP3x restores pheromone dependent signalling in Δras1 (JY1279) cells.
Expression of GFP-Ras1 from pREP3x was used to allow the quantification of Ras1 localisation. Signalling in response to pheromone was analysed to determine whether GFP-Ras1 could support signalling when expressed from
pREP3x in the Δras1 strain JY1279. Assays forβ-galactosidase activity and
average cell volume were performed following growth in a range of pheromone
concentrations (1 nM to 100 μM) in DMM lacking thiamine for 16 h. Cells
expressing GFP-Ras1 and Ras1 from pREP3x displayed very similar dose- response profiles. Cells containing pREP3x-GFP-Ras1 gave a maximal signal
of 12.9 + 0.5 and a pEC
50 of 6.7 + 0.1, compared to a maximal signal of 11.7
+ 0.3 and a pEC
50 of 6.5 + 0.1 in cells expressing Ras1 (Figure 5.3A).
The expression of GFP-Ras1 and Ras1 from pREP3x also supported simi- lar pheromone-dependent increases in cell volume. Cells expressing GFP-Ras1
exhibited a maximal volume of 86.30+ 1.4 fl and pEC
50of 7.6 +0.3. Similarly,
cells containing Ras1 exhibited a maximal volume of 85.8 + 1.3 fl and pEC
50
of 6.8 + 0.3. Cells containing GFP-Ras1 displayed a higher basal cell volume
than those expressing Ras1 (81.6 + 1.9 fl compared to 76.4 + 1.2 fl), possi-
bly indicating a slightly more rounded morphology than cells expressing Ras1 (Figure 5.3B). The expression of GFP-Ras1 from pREP3x causes an increase
in maximal signalling compared to when expressed from theras1 locus (Figure
4.24). This is the inverse effect of that seen with wild-type Ras1, in which con-
stitutive expression causes reduced signalling, due to cell death (Figure 4.42). These data could suggest that the constitutive expression of GFP-Ras1 is not as detrimental to cell viability as that of wild-type Ras1.
***
*
pGFP pGFP-Ras1 pGFP-Ras1Q66LpGFP-Ras1G17VpGFP-Ras1S22N A
B
p
p
p
p
p
Figure 5.4. Analysing the localisation of GFP-Ras1Q66L,
GFP-Ras1G17V and GFP-Ras1S22N using Quimp software
Images of Δras1 cells (JY1279) expressing GFP and GFP-Ras1 fusions from pREP3x were obtained using a Leica SP5 scanning confocal microscope (A). The intensity of fluorescence at the periphery compared to the interior of the cell was determined using Quimp software, and percentage plasma membrane fluorescence was calculated (B). GFP-Ras1Q66Land GFP-Ras1G17V both displayed plasma membrane localisation comparable to GFP-Ras1. GFP-Ras1S22N displayed a lower level of plasma membrane localisation. Statistical significance was determined using a one-way anova with a Tukey multiple comparison post test. Data representative of 10 individual cells + SEM. One aserisk indicates a P-value of 0.05 and three asterisks indicates a P-value of 0.001. The scale bar represents 10 μm.
5.2.4. The localisation of Ras1 is altered in mutations which af- fect nucleotide binding and hydrolysis.
Expression of GFP-Ras1Q66L, GFP-Ras1G17V and GFP-Ras1S22N from theras1
locus revealed no clear change in the localisation of all three mutants com- pared to wild-type Ras1 (Figure 4.45). However, as discussed in the preceding
sections, the expression of GFP-Ras1 fusions from the ras1 locus does not
allow the detailed analysis of localisation. To increase their expression, GFP-
Ras1Q66L, GFP-Ras1G17V and GFP-Ras1S22N were expressed in the Δras1
strain JY1279 from pREP3x. Fluorescence images were then obtained using a Leica SP scanning confocal microscope (Figure 5.4A), and Quimp software was used to determine the level of fluorescence at the plasma membrane (Figure 5.4B).
Both GFP-Ras1Q66L and GFP-Ras1G17V displayed high levels of plasma
membrane localisation. Both exhibited higher mean percentage plasma mem-
brane fluorescence (89.8+2.1 % and 87.4+6.9 % respectively) than GFP-Ras1
(78.0+3.5 %), although this difference was not statistically significant in either
case. GFP-Ras1S22N, by contrast, displayed a significantly lower percentage
membrane fluorescence (59.1 + 6.0 %, P = 0.05). These data could indicate
that the functional interactions of Ras1, which require nucleotide exchange, influence the levels of Ras1 at the plasma membrane.
5.3. Analysing the role of Ras1 C-terminal modification
Ras1 is modified at two C-terminal residues to promote plasma membrane
association (Onken et al. 2006). Cys216, which is contained within a canonical
CAAX farnesylation motif, is the first residue to be modified by the addition of farnesyl group. This event is required for all downstream modifications to proceed. The AAX is then cleaved and the C-terminal cysteine is methyl ester- ified (Eisenberg and Henis 2008). Finally, the Cys215 residue is palmitoylated and the C-terminal modification of Ras1 is complete. The modification of
these residues can be prevented through the use of analogues which sequester the enzymes responsible for these modification, such as 2-bromo-palmitate (Resh 2006), or the more direct approach of site-directed mutagenesis. In the following section, the use of conservative cysteine to serine mutations to pre- vent farnesylation and palmitoylation is discussed. Both the function of these mutants, determined using the assays describe in chapter 4, and their locali- sation, determined using the techniques described in the preceding section, is described.
5.3.1. Preventing lipid modification of Ras1 reduces plasma mem- brane localisation.
N-terminal GFP fusions of the palmitoylation deficient Ras1C215S mutant and
palmitoylation and farnesylation deficient Ras1C216S mutant were created at
the ras1 locus, to allow analysis of the effects of these mutants upon Ras1 lo-
calisation. Integration was performed using the method described previously (Figure 4.28A). Expression was confirmed by immunoblotting using an anti-ras RAS10 antibody, giving a signal at a position consistent with the predicted
size of the fusion (∼ 53 kDa) (Figure 5.5).
In order to determine the effect of altering the C-terminal modification
of Ras1 upon Ras1 localisation, images of cells expressing GFP-Ras1, GFP- Ras1C215S and GFP-Ras1C216S from the endogenous ras1 promoter were ob-
tained. Images were taken using a Nikon E800 epifluorescence microscope fitted with an Andor EM-CCD camera (Figure 5.6). Little plasma membrane
localisation of GFP-Ras1C215Sor GFP-Ras1C216Swas observable. However, the
fluorescence signal was extremely low, preventing clear analysis of the localisa-
tion of either mutant. GFP-Ras1C215S displayed some localisation to internal
structures, which could represent endomembranes. GFP-Ras1C216S appeared
5.3. ANALYSING THE ROLE OF RAS1 C-TERMINAL MODIFICATION 164