The mechanism of protein sorting to the vacuole in yeast was studied both in vitro and in vivo. A series of experiments were performed to reconstitute transport of carboxypeptidase Y (CPY) from Golgi vesicles to vacuoles. In order to investigate this process, microsomes were purified from sec, pep4-3 mutant strains that accumulate inactive proCPY in the Golgi when incubated at the nonpermissive temperature. These were mixed with purified vacuoles isolated from a mutant lacking CPY activity, but containing active proteinases A and B. Transported proCPY is maturated by these proteinases to active form. Experiments indicate that maturation of CPY is due to the correct transport of proCPY from microsomes to vacuoles because:- Firstly, the reaction is temperature sensitive, requires ATP and is stimulated by the addition of soluble factors (S100). Secondly, the addition of proteinase A and B inhibitors to the reaction mixtures has a negligible effect on the maturation process. Thirdly, disrupting the membranes by the addition of Triton X-100 before addition of the proteinase inhibitors, inhibited the maturation of proCPY. Fourthly, the majority of CPY activity was observed in the sedimented fraction of the reaction mixtures rather than supernatant fractions. Lastly, analysis with western blot shows a clear band of mature CPY only in the sedimented fraction of the reaction mixtures with ATP. This in vitro system will be invaluable in investigating the molecular events of vacuolar biogenesis. For in vivo sorting of proteins to the vacuole, a series of experiments were performed that involved the genetic fusion of the CPY promoter and prepro-sequence of CPY to the bacterial Gus (β-glucuronidase) reporter gene. The Gus gene was expressed in yeast with high efficiency and the results of sub-cellular fractionation indicated that the Gus product was distributed in all cell components. Using a centromeric vector gave similar results but with a lower efficiency of Gus expression. Removal of 90bp from Gus, including Gus initiation codon does not completely inhibit Gus expression either in bacteria or in yeast. Fusion of the shortened Gus with the CPY prepro-fragment and expression in yeast led to the correct sorting of the CPY-Gus hybrid protein to the vacuole. This CPY-Gus fusion is potentially useful in the genetic analysis of mutations defective in vacuolar protein sorting.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:328965 |
| Date | January 1989 |
| Creators | Haider, Mustafa M. |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.dur.ac.uk/6495/ |
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