Encuesta realizada a las docentes del centro de educacion inicial “17 de

mRNA localization and cortical ER inheritance are both cell trafficking routes that occur during the process of bud-formation and cell division in S.cerevisiae. After the discovery that both transport courses employ the same locomotion machinery, the Myo4/She3p complex (Estrada et al., 2003), I sought to explore whether the two pathways were functionally linked. Indeed, I found that mutants impaired in ER inheritance like myo4', aux1' and srp101-47ts

simultaneously displayed defects in localization of ASH1 particles (Figure 10). The fact that both the defect in ER inheritance and in mRNA localization are not as prominent in the

srp101-47ts strain as in the other two mutants is most likely due to the fact that this mutant, in contrast to myo4' and aux1' is not a deletion mutant, but a temperature sensitive allele of the wt SRP101 gene. A defect in the two transport routes was expected for the myo4'

mutant as Myo4p is the motor driving both processes. However, the aux1' and the srp101- 47ts mutant so far were only known to have a deficiency in ER segregation (Y. Du et al., 2001; Prinz et al., 2000). Thus, the observation that in addition to ER inheritance defects those mutants are also impaired in mRNA localization provides the first evidence that mRNA localization and cortical ER inheritance are functionally linked and take place in a coordinated manner.

In contrast to our results, a previous study states that mRNA trafficking and ER tubule movement are independent (Estrada et al., 2003). This conclusion was mainly reached by demonstrating that ER segregation is independent of She2p and that in aux1' mutant cells the localized IST2 mRNA can still be detected in the bud by in situ hybridization. At the moment I cannot thoroughly explain this discrepancy but one reason might be the use of different methodologies. Whereas Estrada et al. utilized in situ hybridization in fixed cells, I employed live cell imaging not only for ER structures but also for detection of ASH1 mRNPs by using the MS2-System (figure 10).

The functional correlation between ER segregation and mRNA localization seen in ER inheritance mutants however is consistent with an earlier observation that by in vivo co- imaging, ASH1 mRNPs co-localize and even stay associated with ER tubules (see section 1.5. and A. Jaedicke in (Schmid et al., 2006)). These tubules move from the mother cell to the bud and are required for the segregation of cortical ER during early stages of the cell cycle (Y. Du et al., 2004). Interestingly, another study using microarrays to distinguish pools of membrane associated and cytoplasmic gene products identified ASH1 mRNA as an ER-

Discussion

associated mRNA (Diehn et al., 2000). This result is quite striking as ASH1 mRNA does not encode a membrane or secreted protein but a nuclear transcription repressor and therefore cannot be recruited to the ER via the SRP-pathway (Figure 6). Therefore, this observation supports the aforementioned live cell co-imaging data and the hypothesis of an mRNA-ER co-transport.

In order to explain the discrepancy between Estrada et al. and my data, I employed a live cell imaging system using IST2 as tagged mRNA to be imaged. However, when expressed from its own promoter and even when expressed from a GAL1 promoter the bud localization efficiency for IST2 mRNPs was not the same than for ASH1 mRNPs. For ASH1 in wt cells, localization to the bud occurs in about 92% of the cells. With the IST2 mRNA only a value of 54% was reached in wt cells. As mRNA targeting was already inefficient in wt cells, it was not feasible to use this mRNA for statistical analysis in ER inheritance mutants. However, there is one possible explanation for the low bud-localization rate of IST2 mRNA in live cell imaging. From recent publications it is known that even if targeting of its message is disrupted, there is still some Ist2 protein present in the daughter cell plasma membrane. To some extent Ist2p is transported to the bud independently of mRNA localization. A complex peptide-sorting signal is required for this transport pathway that also works independently of the classical secretory pathway (Franz et al., 2007; Juschke et al., 2004). The fact that for Ist2p, localization is not only mediated by its mRNA but that signals are also encoded on the protein level might be the reason why the transcript is targeted less efficiently to the bud than the ASH1 message, where localization signals are exclusively harboured within the mRNA.

Recently, the finding that mRNA localization and cortical ER inheritance to the yeast bud seem to be coordinated processes was strongly supported by observations of Aronov et al.. Consistent with my data they found that mutations that affect cortical ER segregation to the bud also affect mRNA localization (Aronov et al., 2007). In this study, Aronov and co- workers identified an additional set of 9 bud localized mRNAs encoding polarity and secretion factors in yeast (POL mRNAs). These mRNAs localize asymmetrically to the tip of the emerging bud dependent on their 3’UTRs and the SHE genes and they are bound by the RNA binding protein She2p as demonstrated by IP and RT-PCR (Aronov et al., 2007). Thus, these newly identified mRNAs seem to utilize the same machinery like already described localized yeast messages. The conclusion that mRNA localization and cortical ER inheritance are connected was reached from the finding that POL mRNA localization to the tip of the emerging bud correlated directly with the presence of cortical ER. In analogy to my results Aronov et al. found defects in ER inheritance and mRNA localization not only in the

myo4' strain but also in the srp101-47ts mutant. Additionally, they tested Sec3p, a non- essential subunit of the exocyst complex which mediates targeting of post-Golgi vesicles to sites of active exocytosis (TerBush et al., 1996). Sec3p is thought to act as a spatial

landmark for secretion but was also shown to be involved in ER segregation (Wiederkehr et al., 2003). Similar to aux1', myo4' and srp101-47ts mutants, the SEC3 deletion strain was not only impaired in ER inheritance but also displayed a defect in mRNA trafficking. Finally, not only ASH1 but also the POL mRNAs were shown to associate with ER since they co- fractionated with ER microsomes (Aronov et al., 2007).

Our data in combination with the results from the aforementioned study strongly support the notion of an emerging interplay between the mechanism of mRNA transport and ER inheritance in the yeast bud.

3.2. A connection between mRNAs and membranes: lessons from