Identification and characterisation of Arabidopsis ER accessory proteins

Ferguson, Alison (2012) Identification and characterisation of Arabidopsis ER accessory proteins. PhD thesis, University of Nottingham.

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Abstract

ER accessory proteins are a novel class of endoplasmic reticulum (ER) proteins that facilitate the exit of polytopic membrane proteins from the ER. They are important for the correct targeting of their cognate polytopic membrane proteins to the plasma membrane (PM) and their absence leads to abnormal accumulation of their target in the ER. Until recently, it was not clear if such proteins exist in plants. However, work by Dharmasiri et al (2006) and Gonzales et al (2005) suggest that such proteins exists in plants too. Polytopic membrane proteins such as nutrient transporters, hormone transporters and sugar transporters are a very important class of proteins as they regulate many important physiological and biochemical processes. Better understanding of the targeting of these proteins to the PM is of considerable agronomic interest due to the importance of efficient use of resources in sustainable agriculture.

One of the projects aims is to identify novel ER accessory proteins in Arabidopsis. Using a bioinformatics approach, 40 novel ER resident proteins were identified from a protein localisation database (LOPIT) generated by Dunkley et al (2006) as potential candidates for ER accessory proteins. Genetic, phenotypic and molecular approaches have been used to assess their role as potential ER accessory proteins. A few promising candidates have been identified, one of which AtBPL1 and related family. The AtBPL1 family has similarity to mammalian BAP31 which has been shown to function as an ER accessory protein (Ladasky et al, 2006). To determine if AtBPL1 family plays a similar role in plants a detailed molecular characterisation was carried out, this involved detailed expression analysis using reporter genes and in situ immunolocalisation and characterisation of miRNA lines. Smart screens suggest that BPL1 family members may be involved in the targeting of a nitrate transporter, however its precise target is currently unknown.

A key focus of this present investigation have been on further characterisation of AXR4, which is required for the correct targeting of AUX1 to the plasma membrane (Dharmasiri et al, 2006). AUX1 belongs to a multi-gene family, involving three other members, LAX1, LAX2 and LAX3. Using genetic and cell biology approaches, AXR4 has been shown to be necessary for the correct localisation of at least two other members of this family LAX2 and LAX3. AXR4 mutants show defects in targeting of LAX2 and LAX3 to the plasmamembrane and show weak lax2 and lax3 phenotypes. Co-Immunoprecipitation studies revealed that AXR4 and AUX1 interact directly when co-expressed in insect cells. Finally molecular, bioinformatics and protein modelling approachs were used to probe the function of alpha beta hydrolase domain in AXR4 function. AXR4 appears to be tolerant to amino acid subsitition even at highly conserved amino acids, suggesting that the alpha beta hydrolase domain may not be important for its function.

Item Type:Thesis (PhD)
Supervisors:Swarup, R.
Faculties/Schools:UK Campuses > Faculty of Science > School of Biosciences
ID Code:2472
Deposited By:Mrs Alison Ferguson
Deposited On:14 Nov 2012 15:58
Last Modified:14 Nov 2012 15:58

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