The Sec18 protein (Sec18p) of the yeast <I>Saccharomyces cerevisiae</I> has been identified as a component involved in the vesicular transport of proteins through the secretory and endocytotic pathways. Sec18p is a homologue of the mammalian protein NSF which has been shown, using a number of <I>in vitro</I> transport assay systems and affinity purification procedures, to interact with other proteins in a multisubunit protein complex. This work represents two approaches taken with the aim of identifying proteins that interact with Sec18p in the yeast <I>Saccharomyces cerevisiae</I>. Isolation of protein complexes was first attempted by affinity purification of a tagged version of Sec18p. The protein was C-terminally tagged with a protein A moiety from <I>Staphylococcus aureus</I> containing IgG binding domains. It was hoped that the affinity of protein A for IgG Sepharose could be used to isolate protein complexes that formed <I>in vivo</I> with the Sec18p. Although the fusion construct was shown to be active <I>in vivo</I>, specific complexing proteins could not be isolated due to the large amount of non-specific binding of yeast proteins to the protein A moiety. A second genetical approach was used where the <I>SEC18</I> gene was randomly mutagenised and yeast cells harbouring these mutagenized genes were screened for a dominant negative phenotype. Dominant negative mutant forms of the Sec18p interfere with the normal function of the wild-type protein <I>in vivo</I>. Five such mutants were isolated and classified into two main groups using a number of biochemical and morphological criteria. Class I mutants show a classical secretory mutant phenotype whereas the Class II mutant has a novel phenotype. A number of mutants in which the dominant negative phenotype was suppressed were isolated but the genes responsible for this phenotype could not be identified. It is hoped that alternative strategies can be employed in the future to identify extragenic suppressors of these mutants.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:652121 |
| Date | January 1994 |
| Creators | Harley, Carol |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/20555 |
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