Pidoux, Alison Louise; (1992) Protein targeting in Schizosaccharomyces pombe. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of the work described in this thesis was to develop the fission yeast, Schizosaccharomyces pombe as a model system for studies of the secretory pathway and protein targeting. Two aspects of protein targeting were studied in S. pombe, the characterisation and sorting of the luminal ER protein BiP, and the involvement of ypt/rab proteins in membrane traffic. The homology between BiP proteins from different species was exploited in cloning the gene for S. pombe BiP. The BiP gene was shown to be essential which is consistent with its numerous proposed roles in protein folding and assembly. S. pombe BiP is regulated by a variety of treatments thought to cause an accumulation of misfolded or underglycosylated proteins in the ER, including heat shock. Unusually, S. pombe BiP contains a potential N-linked glycosylation site, which was shown to be partially utilised. The production of antibodies against BiP facilitated an investigation of the structure of the ER in S. pombe. As in mammalian cells the ER of S. pombe has a reticular component. No evidence was obtained for the involvement of microtubules or the actin cytoskeleton in the formation or maintenance of the reticular structure. Amino acids from the C-terminus of S. pombe BiP were attached to the secretory protein acid phosphatase and it was established that ADEL can act as an ER localisation signal on luminal proteins. Signals from other yeast and mammalian species, DDEL, HDEL and KDEL, were also recognised in S. pombe. Members of the ras superfamily isolated in the laboratory using an oligonucleotide representing a conserved region of the GTP-binding domain were characterised. The ypt4 gene represents a novel member of the rab/ypt family. The ypt4 gene is non-essential but in its absence cells contained vacuoles of unusual morphology. An epitope tagged ypt4 protein localised to the surface of vacuoles. These two observations suggest that ypt4 might play some role in vacuole structure or function in S. pombe.

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