When I first deleted SAC1, I used a hygromycin (HYG) marker to replace the SAC1
ORF. At the same time, and in the same diploids SAE2 was also deleted with a
tryptophan (TRP1) marker. After diploid sporulation and tetrad dissection, I noticed
that sac1::HYG cells were extremely unfit and originated small colonies and
sac1::HYG sae2::TRP1 cells were much fitter (Figure 4-11, red versus green).
Although this seemed a very exciting result, sac1::HYG extensive fitness defects
were not suppressed by exo1::LEU2 (or any other gene I deleted without the TRP1
marker). Additionally, SAC1 mutants were shown to have low levels of membrane
phosphatidylserine (PS) (Tani and Kuge 2014). Low PS, in turn, affects tryptophan uptake from the surrounding environment, which leads to tryptophan insufficiency when the cell cannot internally produce tryptophan (Nakamura et al. 2000). I
therefore hypothesized that sac1::HYG cells are sick because they lack tryptophan
and that sae2::TRP1 suppresses sac1::HYG fitness defects due to the TRP1 marker
(and the capacity to synthesise tryptophan) and not loss of Sae2 function.
To test if sac1Δ cells are defective in tryptophan uptake I compared the growth of
sac1::HYG (with and without extra tryptophan supplementation in the medium) to sac1::TRP1 (Figure 4-12). Indeed sac1::HYG (c127) grow worse than sac1::TRP1
Figure 4-11 sac1Δ fitness defects are strongly suppressed by sae2Δ. sac1::HYG
was deleted in the heterozygous diploid vps74::KANMX yku70::HIS3 exo1::LEU2
sae2::TRP1. Diploid was sporulated and spores were separated by tetrad dissection and plates were incubated for 5 days at 23°C before photographing.
Supplementation of sac1::HYG cells with 10 mg/mL tryptophan slightly increased
colony size, but colonies kept growing significantly slower than wild-type colonies.
Importantly, there is no evidence that vps74Δ cells have any issue uptaking
tryptophan neither in the literature nor in my results. For example vps74Δyku70Δ
fitness defects are suppressed by exo1::LEU2 and exo1::URA3, while sac1::HYG
fitness defects could only be suppressed by the presence of the TRP1 marker.
I conclude that the fitness of sac1Δ cells is affected by the TRP1 marker suggesting
defects in tryptophan uptake. Therefore, to avoid fitness defects related to tryptophan
levels, SAC1 should be deleted with the TRP1 marker to allow cells to produce
Figure 4-12 SAC1 deletion affects the cell capacity to uptake tryptophan from the medium. Diploids were generated by deleting SAC1 with a hygromycin (c127) or a tryptophan (c142) marker. Spores were separated by tetrad dissection and plates were incubated for 10 (c127) or 5 (c142) days at 23°C before photographing. Spores from c127 were also dissected onto plates supplemented with 200 µL of a 2.5
4.3. Discussion/Conclusion
The work in this Chapter showed that Vps74 plays an important role maintaining cell fitness and evidence that in Vps74 absence the DDR is activated. A strong genetic
interaction between vps74Δ and yku70Δ, together with a milder interaction between
vps74Δ and cdc13-1, suggested a role for Vps74 at telomeres. VPS74 role at
telomeres, and indeed in DDR, was especially provocative due to the fact that Vps74 is not a nuclear protein and up to date no role for the yeast protein has been
suggested at telomeres. Unfortunately, it was not possible to separate a telomeric role of Vps74 from a role elsewhere in the genome. Actually, the data here presented points towards a more general role of Vps74 in maintaining genome integrity
throughout the genome. This is in agreement with the notion that GOLPH3 (Vps74
mammalian orthologue) is an oncogene since GOLPH3 overexpression could for
example improve cellular fitness.
How Vps74 would contribute to the maintenance of genomic stability is not clear, but mammalian data showed that increased GOLPH3 levels led to cancer (by activation of the mTOR signalling) (Scott et al. 2009). Interestingly, a yeast mTOR orthologue, TORC1 (Tor1 and Tor2), has been associated with telomere maintenance and DDR. For instance, TORC1 inhibition leads to telomere shortening and decreased Yku70 levels (Ungar et al. 2011). Also, rapamycin, a drug that inhibits TORC1, could
strongly suppress cdc13-1 fitness defects after the cells were exposed to 37°C and
put back to 23°C (Klermund et al. 2014). Such effect of rapamycin on cdc13-1 cell
fitness was related to the maintenance of checkpoint proteins in an active
(phosphorylated) state to prevent adaptation/cell division while the cells were at non-
permissive temperatures (Klermund et al. 2014). Interestingly, vps74Δ weakly
suppresses cdc13-1 fitness defects, in agreement with less active TORC1 caused by
VPS74 deletion. In yku70Δ vps74Δ cells, prolonged checkpoint activation would perhaps justify the poor fitness and the reason why deletion of checkpoint proteins suppress the fitness defects. Another more general role for a TORC2 (Tor2) pathway is to maintain genome stability upon mild DSB induction (Weisman et al. 2014). This role is in agreement with a model where Vps74 and Tor2 (and Tor1) collaborate to
maintain genome stability. The fact that vps74Δ cells show mild fitness defects even
at 23°C might suggest slower division/prolonged cell cycle. Prolonged cell cycle could happen if cell checkpoints are being activated by DNA damage.
Vps74 is a cytoplasmic protein, thus a direct role of this protein in the nucleus is unlikely. However, like its human orthologue, it might act as a signal transducer for the DNA damage response. If Vps74 was indeed part of a DNA damage signalling pathway, lack of Vps74 activation could lead to prolonged DNA damage (TORC1 activation for instance), perhaps due to delays in the response or lack of activation of the DDR effector proteins. Bioinformatic analysis of Vps74 phosphorylation sites revealed 39 potential sites (Table 4-2). Indeed Vps74 might be a direct target of Rad53, which also has a small presence in the cytoplasm (Table 4-2) (Smolka et al. 2006). It is therefore possible that Vps74 is important for an effective DDR, being part of signalling cascade. Further work will be required to understand the role of Vps74 in the DDR.
Vps74 has also a small effect on telomere length of yku70Δ telomere defective cells.
Since vps74Δ cells do not have short telomeres, Vps74 role at telomeres is
dependent on Yku70 absence. A possible explanation is that vps74Δ cells
accumulate DNA damage sites and the DDR proteins (including Tel1, for example) are mobilized to those DNA damage sites throughout the genome. A redistribution of some DDR proteins from the telomere to the genome in cells with uncapped
telomeres (yku70Δ cells) might leave the telomere more prone to resection and less
prone to amplification. Such redistribution was already shown to happen upon replication stress and DNA damage, with fluorescent microscopy analysis revealing relocalisation of checkpoint proteins and the Sir complex (Martin et al. 1999; Tkach et al. 2012).
The results here described suggest that Vps74 is involved/affects the DDR (Figure 4-13). I suggest that Vps74 is part of a signalling pathway that responds to naturally occurring DNA lesions (replicative damage or telomere shortening, for example). Lack of Vps74 would therefore delay the repair of the DNA lesions or just prolong the activation of the DNA damage checkpoints (through the TORC1 pathway, for
instance). Lack of Vps74 activity could therefore lead to accumulation of unfixed DNA lesions.
Figure 4-13 Model for a role of Vps74 in the DDR. Natural occurring DNA lesions
(due to replicative stress) or uncapped telomere ends are recognised by the Ku complex. DNA checkpoints are activated and Vps74 is phosphorylated (p-Vps74) by a kinase like Rad53, Yck1/Yck2, Alk1/Alk2, etc. p-Vps74 would then initiate a signal transduction signal, perhaps through the Tor1/Tor2 to aid the DDR. Vps74 might act to amplify the DNA response, without being essential for it.