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Title: Elucidating the function of the suppressor of ppi1 locus 2
Author: Broad, William
ISNI:       0000 0004 7229 6146
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Plastids are a hub of biosynthetic activity, and in different tissues they take on specialised functions and develop into complex structures (e.g., chloroplasts in leaves). The structural and metabolic diversity and dynamism of plastids is dependent upon proteins imported from the cytosol. At the outer-envelope membrane of plastids, such proteins are transported by the translocon at the outer-envelope membrane of chloroplasts (TOC), comprising a protein-conducting channel and receptor proteins. Mutations affecting these TOC proteins cause profound effects to plastid development. To understand more about plastid development and the regulation of the TOC machinery, a screen for second-site suppressors of a mutant lacking the Toc33 receptor, called plastid protein import 1 (ppi1), was previously conducted. In this thesis, the resulting suppressor of ppi1 locus 2 (sp2) mutants were characterised. The SP2 genetic locus was identified, and it was found to affect the development of chloroplasts in a mechanism related to protein import. This was determined to involve a strong negative regulatory effect on the abundance of the TOC proteins, which was reminiscent of the effect of the SP1 locus. SP1 is an E3 ubiquitin ligase that triggers proteasomal degradation of TOC proteins. For this to occur, it is thought that TOC proteins require a mechanism of extraction from the chloroplast membrane, in a pathway that would require a retrotranslocon, as is the case for the degradation of endoplasmic reticulum (ER) proteins by the ER-associated degradation (ERAD) system. As a relative of the Omp85 superfamily of protein transporters, SP2 is here proposed to fulfil the role of retrotranslocon in a hypothesised chloroplast-associated degradation system (ChlorAD). Genetic interaction data suggest that SP1 and SP2 act in a common pathway, while co-immunoprecipitation and in vitro pulldown results reveal a strong physical interaction between SP1 and SP2, as well as an interaction between SP2 and the ERAD-associated ATPase, CDC48. Overall, evidence presented here strongly supports the proposed ChlorAD mechanism.
Supervisor: Jarvis, Paul Sponsor: University of Oxford
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: erad ; arabidopsis ; sp2 ; chloroplast ; toc ; plastid ; sp1