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Effect of the yeast ubiquitination system on proteins accumulated at the entry to stationary phase

Sinclair, Kirsty Ellen; (1998) Effect of the yeast ubiquitination system on proteins accumulated at the entry to stationary phase. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

This study found a beneficial influence of decreased ubiquitin levels on the overexpression of the glucose-derepressed alcohol dehydrogenase, ADH2, in Saccharomyces cerevisiae. Inactivation of the polyubiquitin gene (UBI4) leads to a reduced cellular capacity for ubiquitin-mediated proteolysis, allowing intracellular proteins to accumulate as cells enter stationary phase. Expression of extra plasmid-borne copies of ADH2 enhanced enzyme production sevenfold. As this effect is not protein specific, it may help to improve recombinant protein yields. However, preliminary use of this system to enhance overproduction of two heterologous gene products has proved unsuccessful. This system was also used to overexpress a second homologous G0-induced gene HSP82, encoding the heat shock protein Hsp90. Protein again accumulated to high levels at G0. However, Hsp90 underwent incomplete proteolysis to give several stable fragments, in particular one of 47kDa. The use of domain specific antisera revealed that these cleavage products arise from Hsp82's C-terminal region. Interestingly patients recovering from systemic candidasis may produce high titres of antibodies against a 47kDa immunogen derived from the C-terminus of Candida albicans Hsp90. Further dissection of Hsp82 partial proteolysis revealed that this degradation does not utilise vacuolar proteases but instead requires an intact, functional ubiquitination system. The depletion of ubiquitin in ubi4- cells delays GO Hsp90 turnover. Furthermore, the loss of ubiquitin conjugating enzymes Ubc4 and Ubc5 almost completely prevents Hsp90 partial proteolysis. S.cerevisiae encodes two genes for Hsp90, HSP82 and HSC82. Gene replacement by PCR- generated KanMX4 cassettes produced strains with complete deletions in one or other of these genes. Studies with these strains revealed that whereas Hsp82 is partially degraded, Hsc82 is not and remains 82kDa at G0. This is surprising as the two proteins are nearly identical. Within their 3% disparity includes the replacement of four Hsc82 residues with lysines in the Hsp82 protein. As lysines are sites for ubiquitination, these four Hsp82 residues were mutagenised and their effects on the proteins partial degradation investigated in a Hsp90-free host strain. Mutagenesis of these lysines did not prevent Hsp82 function or partial proteolysis in vivo.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Effect of the yeast ubiquitination system on proteins accumulated at the entry to stationary phase
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Stationary phase; Ubiquitination system
URI: https://discovery.ucl.ac.uk/id/eprint/10097876
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