U531948.pdf (8.17 MB)
Approaches to the study of protein secretion in yeast
thesisposted on 2014-12-15, 10:38 authored by Bhupinder. Gill
Mutants of the yeast Saccharomyces cerevisiae that showed enhanced secretion yield of a mini-proinsulin reporter protein were isolated, following random mutagenesis, via a footprint assay screen. Three such mutants, exhibiting a 2-3 fold increase in secretion yield, were shown to contain recessive mutations in three separate chromosomal genes (ES11, ES14 and ES19). It was not possible to combine these mutations into a single strain as double heterozygous strains failed to produce viable spores. Surprisingly, all three mutants showed reduced levels of the reporter gene mRNA. Expression of a second secretory reporter, wheat -amylase, also showed altered secretory product yield and mRNA levels. However the pattern of effect was different to mini-proinsulin, indicating a specificity of action of the mutations. In contrast, cytoplasmic gene expression was unaffected, with the exception of minor codon containing heterologous genes in esi9 cells, whose expression was blocked. Generally, the three mutants appear to enhance protein secretion by improving efficiency at the initial stage of the secretory pathway probably via the co-translation mechanism. The decreased level of mRNA may result from a direct linkage to the degradation of secretory mRNA, which occurs at an increased rate in the mutants, to its mode of translation.;A second set of mutants showing both enhanced and reduced secretion yields of the Aspergillus niger -galactosidase secreted protein have also been isolated. Again a random mutagenesis approach was used, but this time it was coupled to visual plate assay. These mutants await further analysis.;The final part of this thesis concerns the attempted isolation of SEC gene homologues from Arabidopsis thaliana. Initially this was tried, using a functional complementation approach with sec mutants from Saccharomyces cerevisiae and two A. thaliana cDNA expression libraries but no homologues were isolated. A second approach utilising the PCR and degenerate or homologous oligonucleotide primers, designed to the SEC18 and SEC4 genes respectively, was attempted. Use of the homologous oligonucleotides led to the isolation of possible SEC4 homologous clones from one A. thaliana cDNA expression library but these clones were unable to complement a sec4 mutant. It is possible that these represent a homologue that functions in a slightly different manner in the A. thaliana secretion pathway, as the gross secretion pathway is well conserved between species.
Date of award1997-01-01
Awarding institutionUniversity of Leicester