Physiological and cell biological characterisation of two novel membrane proteins in Arabidopsis thaliana.
MSc(R) thesis, University of Glasgow.
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This thesis describes the characterisation of AtPQL4 and AtPQL6, two members of a hitherto uncharacterized gene family of A. thaliana with six members, AtPQL1-6. As their counterparts in other species and kingdoms AtPQL proteins contain seven transmembrane domains and two copies of the so-called ‘PQ-loop’ domain. AtPQL4 and AtPQL6 show high amino acid sequence identity between each other and they are the closest A. thaliana homologues of the mammalian LEC35/MPDU1 protein, which has been shown to be required for all types of Man-P-Dol dependent glycosylation in the ER.
To address the functional homology between the two AtPQL proteins and MPDU1, the sub-cellular localisation of AtPQL4-GFP and AtPQL6-GFP fusion proteins was investigated. Confocal laser scanning microscopy analysis of Nicotiana tabacum leaf cells expressing AtPQL4-GFP or AtPQL6-GFP showed fluorescence patterns typical for ER. ER localisation of AtPQL4 and AtPQL6 was further confirmed by co-expression with the ER marker, YFP-HDEL.
A second set of experiments employed YFP-fusion proteins of AtSYP121, a plasma membrane SNARE protein, and AtTIP2, a tonoplast aquaporin. Confocal microscopy confirmed plasma membrane/tonoplast localisation of the YFP proteins when expressed on their own in tobacco leaf cells. However, both proteins were found to be retained in the ER when co-expressed with AtPQL4-GFP or AtPQL6-GFP fusion proteins. These new findings point to a role of AtPQL4 and AtPQL6 in protein processing in the ER thereby enforcing previous results from microarray experiments indicating ER-stress in AtPQL4 and AtPQL6 mutants.
Finally, a number of AtPQL4 and AtPQL6 knockout and AtPQL6 overexpressor lines were tested under a variety of environmental stresses to investigate the function of the two AtPQLs at whole-plant level. Low sucrose conditions resulted in growth inhibition of mutants compared to wild type plants. Considering previous findings that (a) AtPQL4 and AtPQL6 are localised in the ER (b) ATPQL mutants show differential expression of genes involved in the unfolded protein response (UPR) and (c) over-expression of AtPQL4 and AtPQL6 impacts on the targeting of other proteins, the observed phenotype could be linked to the unfolded protein response and autophagy that occurs during sugar starvation..
In conclusion, it is proposed that AtPQL4 and AtPQL6 proteins function in retaining membrane proteins for sufficient time in the ER to allow ER-quality control and related processes to take place. Further experiments to investigate such function are discussed.
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