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Title: An investigation of the ypt genes 1, 2 and 5 from Schizosaccharomyces pombe
Author: Craighead, Mark William
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1994
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The ypt genes from Schizosaccharomyces pombe encode low MW GTP binding proteins. These proteins are members of a large family each of which is thought to regulate a specific stage of cellular transport. The aim of this work was to analyse three ypt proteins in an attempt to elucidate their function in vivo. The genes examined were: ypt5, ypt1 and ypt2. The endogenous gene was replaced with a recombinant version linked to a selectable marker. Ypt5p is essential for growth on minimal media and the protein normally receives two geranylgeranyl groups on conserved cysteine residues at its C terminus. The ypt5 gene was replaced with recombinant versions altered at their C termini. Strains containing the mutant ypt5 proteins which only received a single group were viable, however, a doubly mutated protein, which received no geranylgeranyl groups was not capable of sustaining cell growth. Western blotting revealed that both of the singly modified proteins showed less membrane association than the WT protein. The ypt1 gene was randomly mutated prior to recombination and a temperature sensitive strain defective in ypt1 was produced. At restrictive temperatures the Golgi structure was severely disrupted, indicating that the ypt1 protein acts at an early stage in secretion. The strain does not accumulate the secretory protein acid phosphatase although a much reduced rate of secretion was observed. Sequencing revealed that the mutant allele was altered in a residue common to almost all known ypt proteins. The ypt1 gene was specifically mutated so as to encode a protein with the analogous mutation to that found in the ypt1 mutant protein. A strain was created which contained the mutant ypt1 allele and it was found to be temperature sensitive for growth and to accumulate apparently fully glycosylated acid phosphatase, demonstrating that a late stage of secretion was blocked. The defect could be alleviated by expression of both WT ypt2p and mammalian rab8p, strongly suggesting that the two proteins are functionally homologous.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available