The Saccharomyces cerevisiae KEX1 gene product, Kex1p, has been identified and partially characterised to assess its role in processing secreted protein precursors and to define its intracellular location. Kex1p antiserum identified a 113 kDa protein that was absent in kex1-$ Delta$ cells and more abundant in cells overexpressing KEX1. Kex1p was found to be a type I membrane associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kDa. / After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, $ alpha$-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like activity to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. These results provide biochemical evidence, consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins. / Immunological and activity studies indicate that most Kex1p is intracellular and suggests that the enzyme is retained within the secretory pathway. COOH-terminal truncations of the protein indicate that the cytoplasmically exposed domain of Kex1p is responsible for correct localisation of the protein, probably in the late Golgi. / When KEX1 was expressed in Schizosaccharomyces pombe, Kex1p was localised in structures consistent with components of the Golgi. Mammalian cells expressing KEX1 produce a membrane associated activity that is not detected in the medium. In immunofluorescence studies on mammalian cells, Kex1p was localised to the ER and Golgi but not to the plasma membrane. Kex1p in such cells was responsible for completing the processing of the neuropeptide, $ gamma$-lipotropin. This in vivo processing of $ gamma$-lipotropin by Kex1p demonstrates a significant functional homology of the basic prohormone processing machinery in yeast and neuroendocrine cells.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.74650 |
| Date | January 1990 |
| Creators | Cooper, Antony |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001224828, proquestno: AAINN67741, Theses scanned by UMI/ProQuest. |
Page generated in 0.0043 seconds