The role of Cdc48 adapter proteins Npl4 & Ufd1 in chloroplast associated protein degradation

The vast bulk of the chloroplast proteome is imported post-translationally through protein translocation complexes called TOCs (translocon at the outer envelope of chloroplasts). The regulation of such an import system offers a key mechanism to impact chloroplast metabolism and plastid development, both through the modulation of the proteome. In response to biotic, abiotic, and developmental cues, the TOC proteins are targeted by the ubiquitin-proteasome system for degradation. Membrane-embedded TOC proteins must first be extracted from the outer membrane before degradation by the cytosolic UPS. This is accomplished by retrotranslocation through a protein channel, with the motive force provided by an ATPase called CDC48 — this pathway has been termed CHLORAD (chloroplast associated protein degradation). CDC48 functions in a variety of cellular processes at different subcellular locations. The localisation and activity of CDC48 is determined by a wide range of adapter proteins. In UPS processes, CDC48 is typically recruited by a heterodimer of NPL4 and UFD1. There are two NPL4, and four UFD1 isoforms in the model higher plant Arabidopsis thaliana. To examine their potential role in CHLORAD, I performed bioinformatic analysis on protein sequences and predicted structures; interaction and localisation experiments, using microscopy and immunoprecipitation; and finally conducted a mutant analysis of knockout and overexpressing lines, looking at developmental and stress responses. Three UFD1 and two NPL4 proteins can participate within the CHLORAD system by recruitment of CDC48. While the proteins are nucleocytosolic, interactions with TOC proteins were sustained at the chloroplast outer envelope membrane. There appears to be a degree of functional redundancy between sets of the isoforms; this is supported by phylogenetic reconstruction showing isolated clades in the Arabidopsis family. Few phenotypes were observed in the analysis of single mutants. One significant one on TOC protein abundance may be the product of a dominant negative mutation generated through a translational fusion with an epitope tag. The functional significance of NPL4 and UFD1 in CHLORAD requires much additional consideration in the future, with particular emphasis on the analysis of additional genetic lines. Phenotypes may be masked by functional redundancy between NPL4 and UFD1 isoforms, so the investigation of appropriate multiple mutants would be highly informative. Additionally, analysis of crosses to plastid protein import mutants would determine if there are suppressive genetic interactions, which would support a function in CHLORAD. Additional focus should be placed on the generation of domain deletion mutants to validate bioinformatic predictions of function — Ufd1C should be the prime candidate in this regard, to assess how interactions are sustained with a remarkably unique predicted structure. Finally, consideration should also be given to the potential role in biotic stress tolerance, as both CHLORAD and CDC48-NPL4-UFD1 have independently understood contributions to the immune system.

Keywords: chloroplasts
protein import
Npl4 and Ufd1
ubiquitin proteasome system
CHLORAD
Advisor: Jarvis
Van der hoorn, R