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Title: Golgi protein localisation in Schizosaccharomyces pombe
Author: Williams, Hazel Patricia
ISNI:       0000 0001 3569 0572
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1999
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The aim of the research was to identify Golgi targeting motifs in the galactosyltransferase Gma12p in Schizosaccharomyces pombe. For this study it was tagged at its C-terminal end with the Green Fluorescent Protein (GFP) allowing mutations that cause fusion protein mislocalisation to be easily identified by confocal microscopy. All regions of the enzyme were studied for a possible role in localisation. Deletion analysis was carried out on the lumenal domain. This showed that the catalytic domain was not required for correct Golgi localisation. Deletion of parts of the stem region then showed that at least seventy three amino acids from this region were required. The role of the TMD was also explored. Initially it was investigated what other TMDs could substitute for that of Gmal2p and still confer Golgi localisation. Therefore, the TMD was swapped with those of three homologues of Gma12p, Gth1p, Gth3p, Gth5p. All three conferred Golgi localisation as measured by fluorescent microscopy. However, these four TMDs show little sequence homology and vary in length from approximately 22 to 28 residues. Therefore the TMD was specifically altered in length by the addition or deletion of three hydrophobic residues. The lengthening of the TMD by three residues caused substantial mislocalisation of the fusion protein. This mislocalisation was also seen when either or both of two threonine residues within the TMD were replaced with leucine. These mutant fusion proteins were localised to an endoplasmic reticulum (ER) compartment as shown by colocalisation with the ER chaperone BiP. The fusion proteins were shown to be folding correctly, making it unlikely that they are retained in the ER by quality control. Therefore, I propose that the two threonines in the TMD mediate a protein protein interaction that is required for efficient transport of Gma12p from the ER to the Golgi apparatus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry