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Sss1p is an essential component, along with Sec61p, of the protein conducting channel (PCC) in the ER of the yeast Saccharomyces cerevisiae. It belongs to a family of proteins termed tail-anchor (TA) proteins. The TA consists of a single hydrophobic sequence at the carboxyl-terminus which anchors the protein to the membrane in a Type II (N_cytoplasm-C_lumen) orientation. TA proteins are targeted to their membranes of function through an uncharacterized SRP-independent, post-translational mechanism. The targeting mechanism and function of Sss1p are not known. In this thesis, results will be presented from targeting and functional studies of Sss1p. Sss1p is predicted to contain an ER targeting signal similar to mammalian VAMP-1A. Disruption of this putative signal caused incomplete mislocalization of Sss1p to the mitochondria which did not affect yeast growth. Mutations in the TA of Sss1p had numerous effects. Yeast expressing these mutants showed diminished growth, a defect in co-and post-translational translocation, inefficient ribosome binding to Sec61 p and the mislocalization of translocon components from light membranes (predominantly ER) to heavy membranes (predominantly mitochondria). It is argued that mutations in the TA of Sss1p disrupt the function of the protein, subsequently leading to the general defects listed above. Two possible functions for Sss1p are proposed: Sss1p is involved in forming the signal sequence binding pocket of the translocon and/or is essential for the integrity of the PCC. / Thesis / Master of Science (MS)

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A range of structurally diverse, novel antimicrobial agents - non-polymerizable, polymerizable and polymeric - have been synthesized and tested for antimicrobial activity against the yeast strain Saccharomyces cerevisiae EL 1 in orange and apple juice media. The aim of this work was to provide attractive candidates for use in liquid food preservatives. The acrylamido thiazolyl monomer 67 (Scheme 1) was prepared but tested as inactive. Attempts to prepare the novel thiazolylsulfanilamide monomer 69 (Scheme 2) met with failure. The non-polymerizable antimicrobial agents benzofuroxan 71 (Scheme 3), nonyl 3-amino parabens 78 (Scheme 4) and benzokathon 99 (Scheme 6) were prepared and tested for antimicrobial activity. Benzofuroxan 71 and, in particular, the parabens compound 78 displayed significant activity, whereas benzokathon 99 showed little activity. Polymerizable analogues of compounds 71, 78 and 99 would make useful targets towards the development of polymeric antimicrobial agents. Attempts to prepare the chloropropyl kathon 93a and the bromoethyl kathon 93b (Scheme 5) met with failure. In principle, the kathons 93a and 93b could be modified, through relatively straightforward chemistry, to polymerizable analogues. The triethyl, tri-n-butyl, tri-n-octyl and triphenyl polymerizable phosphonium salts 105a-d (Scheme 7) were prepared as m/p-isomeric mixtures. The pure p-isomeric analogues 111b-d were prepared also. Only the tri-n-octyl phosphonium salt l05c displayed antimicrobial activity. This result supports the general finding that cationic biocides with long alkyl chains are more active than those with shorter chains. The tri-n-butyl phosphonium salt monomer 105b was polymerized. The resulting homo-polymer 112b (Scheme 8) was found to inhibit yeast growth more than the corresponding monomer 1OSb. This result supports the general finding that polymeric biocides are more active than the corresponding low molecular weight compounds. The diiodomethyl sulfone 125 (Arnical) and the analogous monoiodo species 126 (Scheme 10) were prepared and tested for antimicrobial activity. Compounds 126 and, in particular, 125 displayed very potent activities. A polymerizable analogue of Arnical would make a useful target towards the development of polymeric antimicrobial diiodomethyl sulfones. The chemical breakdown of Amical 125, in chloroform, to molecular iodine was investigated by UV spectrophotometry. The rate of iodine production was found to be accelerated by ultraviolet light and to be decelerated by darkness. As a result of this discovery, a photochemical mechanism was proposed for the degradation. Further studies revealed that the rate of iodine production was inversely proportional to the Amical solution concentration. The monoiodo analogue 126 of Amical was also observed to break down in chloroform to produce iodine. However, iodine production resulting from the degradation of 126 was not as great compared with that of Amical. As a result, it was concluded that iodine production increases with the degree of iodonation. This conclusion was supported by UV spectrophotometric studies of the 'model' compound, iodoform (CHI3) - for solutions of identical concentration, iodine production increased in the order: 126 < 125 < CHI3. The chemical modification of Amberlyst 15 and Dowex 50-X8 sulfonic acid ion exchange resins 138 was conducted, in parallel, with the aim of producing a polymersupported diiodomethyl sulfone 142a (Scheme 12). Analytical evidence suggested that the transformation 138-142a was more successful on the Amberlyst resin. Despite this, however, only very low iodine loadings were obtained for the Amberlyst resin 142a. The Amberlyst- and Dowex-derived resin beads 138, 139, 141 and 142a were tested for antimicrobial activity in the 'dry' and 'wet' states using plate assay and liquid assay techniques respectively. Although some beads were active, and others were not, the microbiological data was too inconsistent, in general, to form any concrete conclusions regarding activity trends. The series of novel polymerizable nicotinate quaternary ammonium salts 148a-i (Scheme 13), with esters of different chain length and structure, were prepared and tested for antimicrobial activity. A selection of the analogous isonicotinate compounds 149 were also prepared and tested. It was found that an increase in ester chain length lead to an increase in activity, with the Cg, C9 and C12 nicotinate quats (l48g, 148h and 148i respectively) testing as the most active. In addition, it was found that the straight -chain compounds were more active than their branched-chain counterparts (e.g. 148e was more active than 148f). The position of the ester group on the pyridinium ring (c.f. 148 and 149 series) did not appear to significantly influence antimicrobial activity. The octyl nicotinate quat 148g was polymerized with the aim of producing a water-soluble polymeric pyridinium salt. However, the resulting homo-polymer 150 (Scheme 14) was insoluble and, as a result, was not tested for antimicrobial activity. A selection of the analogous isonicotinate compounds 149 were also prepared and tested. It was found that an increase in ester chain length lead to an increase in activity, with the Cg, C9 and C12 nicotinate quats (l48g, 148h and 148i respectively) testing as the most active. In addition, it was found that the straight -chain compounds were more active than their branched-chain counterparts (e.g. 148e was more active than 148f). The position of the ester group on the pyridinium ring (c.f. 148 and 149 series) did not appear to significantly influence antimicrobial activity. The octyl nicotinate quat 148g was polymerized with the aim of producing a water-soluble polymeric pyridinium salt. However, the resulting homo-polymer 150 (Scheme 14) was insoluble and, as a result, was not tested for antimicrobial activity. The m/p-isomeric version 151 of the quat 148g was co-polymerized, separately, with acrylamide and 2-hydroxyethyl methacrylate (Scheme 15). It was hoped that co-polymerizing 151 with a hydrophilic monomer would produce a water-soluble pyridinium salt co-polymer. However, both the co-polymers 153 and 154 were insoluble in water and, again, testing of these materials was not pursued.

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