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1. The GDP-bound rabp is complexed to GDI in the cytoplasm. The GDI-rabp complex interacts with a GDS on the donor compartment/ transport vesicle, causing a GDP- GTP exchange. 2. The GTP-bound rabp associates with a complex on the transport vesicle. The complex may include a v-SNARE.

3. At the acceptor a v and t SNARE pair interact. A GAP on the membrane stimulates the rabp to hydrolyse its GTP. The energy released being used to lock the vesicle in a fusion competent state. 4. GDI removes the GDP-bound rabp from the membrane and recycles it to the donor membrane.

The vesicle would then bud from the donor and travel to the target membrane. The complex containing the ypt/rab protein would then be involved in docking the vesicle to the correct acceptor compartment. Presumably the v-SNARE on the vesicle will bind the t-SNARE on the target membrane. The ypt/rab protein would act as a second level of control on specificity with it being activated by a GAP activity on the membrane to hydrolyse its bound GTP to GDP. This allows for a two-step recognition process: the v and t-SNARE pair, and the rab protein with its specific GAP. The dual recognition model explains the need for specific rab proteins, SNAREs and GAPs. The energy of hydrolysis would lock the vesicle in a fusion competent state. Fusion would continue with NSF etc all playing a role. After hydrolysis the rab would be in a GDP-bound state and could therefore be removed from the membrane by GDI and the cycle would continue.

1.7 Schizosaccharomyces pomhe

The fission yeast S.pombe was first isolated from beer in Africa. S.pombe differs quite markedly from the primitive budding yeast S.cerevisiae. One of the most notable differences is in the growth cycle of the two yeasts. S.cerevisiae grows by forming a bud which grows in size to eventually form a daughter cell which separates from the parental cell. S.pombe is a fission yeast and multiplies in a manner which is more similar to higher eukaryotes. The S.pombe cell is cylindrical, about 3.5|Lim in diameter and 7|im in length. Growth occurs throughout the cell cycle with extension occurring at one end of the cell at any particular time, for a review of S.pombe cell growth see (Johnson, et al., 1989). When the cell has doubled in length a septum starts to form at the centre of the cell which increases in size eventually separating the two daughter cells.

1.7.1 The Golgi complex

S.pombe not only differs from S.cervisiae in its growth cycle but also in its morphological and biochemical makeup, both of which are closer to that seen in higher eukaryotes. As stated earlier the Golgi in S.cerevisiae consists of separate cistemae whereas in mammalian cells stacks are observed. The Golgi complex in S.pombe is readily observable. The number of cistemae in the stack varies but on average there are three per stack with twenty such stacks per cell (Ayscough, 1993). The number of stacks increases during the cell cycle. There is no morphological evidence of any asymmetric distribution of enzymes within the stacks, with electron microscopy indicating that galactosyltransferase is

distributed throughout each stack (Ayscough, 1993). Analysis of secretory proteins has revealed that, unlike S.cerevisiae, which only adds mannose to carbohydrate side chains, both galactose and mannose are added to the sugar groups of secretory proteins as they pass through the Golgi complex (Chappell and Warren, 1989).

1.7.2 S.pombe genetics

Although S.pombe is only distantly related to the budding yeast S.cerevisiae it does have the same features that makes S.cerevisiae such a genetically tractable organism. S.pombe exists normally as a haploid organism but nutrient deprivation will result in the formation of diploid zygotes which will normally sporulate to form haploid progeny (Egel, 1989). Strains can be forced to remain diploid by depriving them of specific nutrients the haploids cannot synthesise. The availability of both haploid and diploid strains makes S.pombe a very useful organism in terms of allowing gene disruptions and in the production of conditional mutants.

Recently a large number of genetic techniques, including transformation and the construction of expression vectors have successfully been applied to S.pombe (Moreno., et al., 1990). S.pombe has now proved to be an excellent model for higher eukaryotes, for example the cell cycle in S.pombe has been studied in great detail (Fantes, 1989).

1.7.3 YPT genes from S.pombe

Until recently only two YPT genes had been identified in S.cerevisiae whereas in mammalian cells a large number of rabs had been identified. A number of groups, including our own, attempted to locate ypt genes in S.pombe. It was reasoned that the proteins identified could well be models for those already identified in mammalian cells. A total of six genes were rapidly identified by various genetic screening methods (Fawell, et al., 1989; Fawell, et al., 1990; Haubruck, et al., 1990; Hengst, et al., 1990; Miyake and Yamamoto, 1990). Sequencing revealed all of the genes discovered, except for ypt4, were homologues of previously identified mammalian genes (Table 1.1).

1.8 AIM OF PROJECT

At the beginning of this work our group had recently identified a number of ypt genes from S.pombe which we now wished to characterise. My initial aim was to establish a system which would allow the endogenous ypt genes to be replaced by recombinant copies linked to a selectable marker. The system would then be used to generate conditional mutants of the ypt genes by either specifically or randomly mutagenising the recombinant gene prior to replacement. The resultant strains would then be examined both biochemically, by examining the carbohydrate modifications of secretory proteins, and morphologically by microscopic analysis. By this route we hoped to identify which stage of transport each ypt protein was operating at. We also wished to use mutant strains to discover if they could be rescued by the expression of the mammalian hom ologue i.e. would overexpression of rabS protein rescue a strain with a defective ypt2 gene.

As said we wished to produce stains of yeast containing ypt genes with specific point mutations. Doing this would allow us to correlate in vitro results with results in vivo. In particular we wished to investigate the post-translational modifications received by the ypt proteins and the effect of these modifications, or lack of them, on the protein in vivo.

Chapter 2

Materials and