BENEFICIOS ECONÓMICOS

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Fig. 3.6

Cells were grown in liquid at 30^C and either left at this temperature or shifted to 16«C for 12 hours. (A) 556 3Q0C (B) CSSypt5 3Q0C. (C) CSSypt5

160C. (D) CSSypt5: YPT5 16«C. CW- cell wall. G- Golgi. M- mitochondria. N- nucleus. S- membrane structures. V- vacuole. Bar 0.5p.m.

3 .6 D iscu ssio n

The initial aim of this work was to develop a system which would allow the replacement of the endogenous ypî5 gene in S.pombe. In an attempt to do this the yptS gene and flanking sequences were cloned into a plasmid. The S.pombe ura4 gene was also cloned into the plasmid 40bp downstream of the yptS coding sequence. Purified insert from the plasmid was then used to transform a haploid strain of S.pombe. The rate of homologous recombination was then determined by PCR and later confirmed by Southern blotting. The rate o f homologous recombination was found to be approximately 10%. The system was developed with the intention of producing conditional mutants o f the yptS gene. It had been intended to mutagenise the insert prior to transformation in the hope that we could subsequently select for conditional mutants. However, it was felt for reasons outlined in section 3.2.1 that the rate of homologous recombination was too low to allow the rapid production of conditional mutants of the yptS gene. We believed that it would be potentially more productive to replace the endogenous gene with recombinant copies which had been specifically mutagenised, as a high percentage of the homologous recombinants recovered, approximately 1 in 10 of all viable transformants, would contain the altered yp tS gene. The exact fraction of recombinants which would contain the altered yptS allele would vary depending on which part of the ypî5 gene was modified i.e. those parts of the yptS gene closest to the ura4 gene would be selected for with the greatest efficiency.

As I discussed in the introduction all ypt/rab proteins have a conserved cysteine motif at their C terminus. It has been shown that the cysteine residues are modified with Geranylgeranyl (GeGe) moieties (Khosarvi, et al., 1991; Kinsella and Maltese, 1991). The ypt proteins from S.pombe were also found to have cysteine residues at their C termini (Miyake and Yamoto, 1990; Haubruck, et al., 1990). It had been previously shown that y p tlp , yptSp and yptSp from S.pombe re c e iv e d geranylgeranyl groups when they were expressed in vitro (Newman, et al., 1992). It had also been shown that the addition of the GeGe groups was necessary for the membrane association of the S.pombe proteins in vivo (Newman, et al., 1992).

In an attempt to further characterise the modifications that the ypt5p was receiving a number of specifically mutated yptS cDNAs were created by Chris Newman in Dr A. Magee's laboratory. The ypt5 alleles were altered at their extreme C terminus so as to encode either -CSC (control), -CSS, -SSC or -SSS. Dr Magee's group wished to study the modifications that the mutant proteins received. In parallel we were

The mutant cDNAs were used to create mutant genes as described in section 3.2.2. The mutant genes linked to the selectable marker ura4 were then used to transform a diploid S.pombe strain. The replacement experiment was initially attempted on the haploid 556 strain, in retrospect this was not the best strategy as only a positive result was possible i.e. a non-functional allele would give no homologous recombinants but of a lack of homologous recombinants would not prove that the protein encoded by the allele was non-functional. After transformation the diploid strains were screened and homologous recombinants identified both by PCR and by Southern blotting.

The diploid strains were next analysed by Random Spore Analysis which revealed that the two singly altered ypt5 proteins yptCSSp and ypt5SSCp were both functional i.e. strains containing the mutant alleles as the sole copy of yptS were viable on minimal media. However, the doubly mutated protein ypt5SSSp was not functional. Work by our collaborators indicated that ypt5SSSp was not modified in vitro or in vivo whilst the singly mutated proteins ypt5CSSp and ypt5SSCp received only 50% GeGe groups per cysteine residue in comparison to the WT ypt5p. It was concluded therefore that the ypt5p receives two GeGe groups, as is seen for the rabSAp (Farnsworth, et al., 1991), and that the GeGe groups were essential for the function of the ypt5p. The results obtained in S.pombe cells were similar to those reported for both yptlp and sec4p from S.cerevisiae (Molenaar, et al., 1988; Walworth et al., 1989). Both groups reported that removal of C terminal cysteine residues resulted in a non-functional protein which was completely cytoplasmic. We then went on to analyse the localisation of the various ypt5 proteins both in transfected COS cells and in S.pombe. Our collaborators found that the WT ypt5p demonstrated 50% membrane association, both of the singly altered proteins were less than 10% membrane associated whilst the doubly mutated ypt5SSSp was completely cytoplasmic (Giannokouros, et al., 1993). Western blotting of S.pombe cell extracts confirmed that the point mutants ypt5SSCp and ypt5CSSp were less membrane associated than the WT ypt5p. In order to quantify the distribution of mutant proteins in S.pombe the experiment shown in Fig. 3.5 will have to be repeated in the presence of purified ypt5 protein to demonstrate that the observed bands are all specific. Also controls will have to be carried out to ensure that the ypt5 proteins are all being recovered to the same extent. For example the proteins were precipitated using the methanol/ chloroform method (Wessel and Flugge, 1984) and it would be desirable to repeat the experiment with a different precipitation procedure to ensure that the all of the various forms of the ypt5p were being recovered equally. Furthermore one could precipitate protein from total cell

extracts and compare the level of ypt5p with that found in the membrane and cytoplasmic fractions to ensure that the recovery levels are equal in the different strains.

I next went on to analyse the growth of the mutant strains at various temperatures. This analysis was done by visual inspection and therefore any conclusions on the growth properties will have to be confirmed by quantification. It was observed that the two mutant strains, termed SSCypt5 and CSSypt5, were capable of growing at 30®C, 37®C and 18®C. It was initially observed that the cells displayed a growth deficiency at 14®C in comparison to WT 556 cells, however, if the experimental procedure was altered such that the cells placed at 14°C were not in stationary phase then it was seen that the strains were capable of growing. Also it was observed that the SSCypt5 strain would no longer grow after storage at 4°C. In the future it would be desirable to repeat the growth analyses at different temperatures in a much more rigorous manner, with regular cell counts and with the experiment being repeated on several occasions. The results obtained seem to imply that the mutant cells may lose viability as they enter stationary phase. This can be analysed by plating out cells from cultures at different stages of growth eg when the cells are growing logarithmically and then as they enter stationary phase and after prolonged stationary phase. The cell numbers can be counted prior to plating onto fresh media, or dilution into fresh media, and viability can be determined by for example counting the number of colonies that appear on the plate.

Electron microscopy revealed the accumulation of large membrane enclosed structures in CSSypt5 cells which had been grown to stationary phase and then diluted into fresh media and incubated at 16®C. These structiu*es were not seen in the same strain transformed with WT yptS cDNA indicating that their presence was due to the defective ypt5 protein. If it is shown that these structures arise as a direct result of the recombinant ypt5 proteins then the most compelling explanation, for their production, is that they arise as a result of a defect in endocytosis. It will be interesting to examine the morphological effects in the SSCypt5 strain to determine whether this strain also forms these structures. It will also be interesting to analyse cells which have been incubated in stationary phase for varying lengths of time.

Assuming that the structures are formed due to a defect in endocytosis there are two possible explanations for their production (Fig. 3.7). It is possible that a non­ functional ypt5 protein would somehow cause a block in budding from a compartment, presumably indirectly as a result of sequestering components which are required for budding. However, I believe that a more plausible explanation, bearing in mind that ypt proteins are thought to regulate fusion, is that the lack of functional ypt5 protein may allow aberrant fusion by not blocking the association of the wrong v and t SNARE pair.

At the moment work is going on to develop an endocytosis assay in S.pombe when there is such an assay we will then be able to ascertain whether the mutant ypt5 proteins are deficient in endocytosis or not. Using the gene replacement system it may be possible to engineer strains which are cold sensitive due to a mutation in the ypî5 gene. If this could be achieved it would greatly enhance our ability to study the role of ypt5 in vivo.

Fig. 3.7 Effect of mutant yptSp