S-acylation, commonly known as S-palmitoylation, is a reversible posttranslational lipid modification in which fatty acid, usually palmitic acid, covalently attaches to specific cysteine residues of proteins via thioester bonds. Palmitoylation enhances the hydrophobicity of proteins and contributes to their membrane association. It plays roles in protein trafficking, signalling, protein-protein interaction, protein stability and other important cellular functions. A family of Protein S-acyl Transferases (PATs) is responsible for this reaction. PATs are multi-pass transmembrane proteins that possess a catalytic Asp- His-His-Cys cysteine rich domain (DHHC-CRD) of ~50 amino acids. In Arabidopsis there are at least 24 such DHHC-CRD containing PAT proteins and they are named as AtPAT01 to AtPAT24. The function of only 2 AtPATs, AtPAT10 and AtPAT24 were studied in some detail, and a recent survey showed the ubiquitous expression pattern and different membrane localization habit of all 24 AtPATs. However, the biological function of the remaining 22 AtPATs in Arabidopsis was not reported when I started my project. Therefore, we carried out an initial screen of all the available T-DNA insertion lines of the 22 Arabidopsis PATs and identified transcriptional null mutants of 18 of the AtPATs. Among them, the k/o mutant plants of only 3 genes showed significantly altered phenotypes compared to wild-type Arabidopsis, and the mutants are named as atpat14, atpat21 and plp1(PAT-like Protein 1). This project aims to characterize these three putative PATs in details in terms of their PAT activities, catalytic domains, expression patterns, subcellular localizations and biological functions. AtPAT14 was proved as a PAT by yeast complementary and in vitro auto-acylation assays. Mutagenesis studies clearly demonstrated that the cysteine residue in the DHHCmotif is essential for the enzyme activity of AtPAT14. Transgenic Arabidopsis plants expressing AtPAT14-GFP were observed and it was shown that AtPAT14 is predominantly localized at the Trans-Golgi. The phenotype was observed in both atpat14-1 and atpat14-2 mutant lines and this showed that the leaves of both lines were aging much faster than the WT. Analysis of the levels of different phytohormones revealed that the mutant leaves contained much higher salicylic acid (SA) than the WT. This coincided with the increased transcript levels of genes involved in SA biosynthesis and signalling. Therefore, AtPAT14 mediated protein S-acylation plays important roles in leaf senescence via the regulation of SA biosynthesis and signalling pathways. AtPAT21 was also confirmed as a PAT and the DHHC its functional domain by similar approaches as for AtPAT14. The plasma membrane (PM) localized AtPAT21 plays essential roles in both male and female gametogenesis. As such, loss-of-function by TDNA insertion in AtPAT21 leads to the plant being completely sterile. Therefore, AtPAT21-mediated S-acylation of proteins(s) plays important roles in the reproduction of Arabidopsis. AtPLP1 (PAT-like Protein 1) contains the signature DHHC-CRD. However, it does not rescue the growth defects of akr1, pfa3 and swf1, the 3 yeast PAT mutants used in enzyme activity assays of other known PATs from plant and animals. Further, the cysteine residue in the DHHC motif was not essential for the function of AtPLP1 as mutated variant containing serine in place of cysteine of the DHHC motif can still rescue the growth defects of atplp1-1. Seedling establishment of atplp1-1 was impaired without external carbon source. This is because the efficiency in converting the seed storage lipid to sugar in the mutant is much lower than WT due to the defective β-oxidation process involved in the degradation of free fatty acids released from lipid during post-germinative growth. In addition, atplp1-1 seedlings are also de-etiolated in the dark, and this was coincided with more cytokinin (CK) and less active gibberellin (GA) related pathway in the mutant. Other defects were also found in atplp1-1, such as hypersensitive to abscisic acid (ABA) and sugar during seed germination and abnormal shoot apical meristem (SAM) in older plants. Therefore, protein S-acyltransferases play distinct and diverse roles throughout the life cycle, from seed germination, seedling growth to seed production in Arabidopsis. This is most likely through the palmitoylation of an array of proteins they modify. Hence, our results provide vital clues for future studies on the molecular mechanism as to how AtPATs operate in plant.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:715280 |
| Date | January 2017 |
| Creators | Li, Yaxiao |
| Contributors | Scott, Roderick ; Doughty, James |
| Publisher | University of Bath |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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