Genetic and molecular characterization of the regulation of chloroplast protein import in Arabidopsis thaliana

Thesis

Plastids are a unique group of interconvertible organelles which are descended from prokaryotic endosymbionts. The most prototypical member of the group is undoubtedly the chloroplast, which carries out photosynthesis and many other essential biosynthetic processes. Within the course of evolution, over 90% of the retained ancestral protein-coding genes have been transferred into the nuclear genome. The encoded chloroplast-destined proteins must therefore be imported as precursor proteins from the cytosol, through the import apparatus comprising the TOC/TIC complexes. Over the last few years, a proteolytic system called the Ubiquitin-Proteasome System (UPS) has been recognized to target TOC proteins for degradation. The dynamic regulation of the import pathway enables plastids to respond to environmental and developmental cues. A recent discovery demonstrated that the ubiquitinated membrane-embedded TOC proteins can be extracted through a pathway called chloroplast-associated protein degradation (CHLORAD) before degradation by the cytosolic 26S proteasome. In CHLORAD, SP1 acts as the E3 ligase, SP2 functions as an exit channel, and the ATPase Cdc48 provides the motor force for extraction.

Cdc48 functions in a variety of cellular processes and its activity depends on a wide range of cofactors. The ubiquitin regulatory X (UBX) domain-containing proteins constitute by far the largest family of Cdc48 cofactors. PUX10, a member of the plant UBX domain-containing protein (PUX) family, is an integral chloroplast outer membrane protein that contains a UBX domain and a ubiquitin-associated (UBA) domain that interact with Cdc48 and ubiquitin, respectively. Although pux10 mutant Arabidopsis thaliana seedlings in general appeared phenotypically normal, PUX10 expression levels influenced the rapid degradation of over-accumulated TOC proteins under certain circumstances, such as senescence or abiotic stress. Thus, I propose that PUX10 is involved in the CHLORAD pathway, mediating the recruitment of Cdc48 from the cytosol to the chloroplast surface, and promoting Cdc48 binding to polyubiquitinated TOC proteins for degradation.

In a parallel genetic strategy to understand chloroplast protein import regulation, I studied a new suppressor of the plastid protein import 1 (ppi1) mutation in Arabidopsis called suppressor of ppi1 locus 3 (sp3). Preliminary characterization suggested that the SP3 encodes the GIGANTEA protein which regulates photoperiodic flowering and negatively regulates chloroplast biogenesis. Further investigation of the functional interactions of SP3 with CHLORAD and TOC components will be required to fully understand the nature of these relationships.

Authors: Li, N

• DOI: 10.5287/ora-gpem4bbej
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: chloroplast; CHLORAD; PUX10; UBX; chloroplast protein import; Cdc48
• Subjects: Plant molecular biology