Pattison, Richard John
Characterisation of the PQ-loop repeat membrane protein family in arabidopsis thaliana.
PhD thesis, University of Glasgow.
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This thesis describes the characterisation of the PQ-loop repeat family, a novel family of membrane proteins from Arabidopsis thaliana. The family consists of six members (AtPQL1-6) all predicted to contain seven transmembrane segments and two copies of the PQ-loop domain. The repeated PQ-loop domain is a well conserved feature present in several eukaryotic proteins. However, the functional significance of the domain remains to be determined. Although most PQ-loop repeat proteins are of unknown function some have been characterised from non-plant species. Those studied include CTNS - a lysosomal cystine transporter from humans, STM1 - a putative G-protein coupled receptor from Schizosaccharomyces pombe, ERS1 - a Saccharomyces cerevisiae protein isolated as a suppressor of a trafficking mutant and LEC35/MPDU1 - an endoplasmic reticulum protein from mammals involved in the utilisation of dolichol monosaccharides during glycosylation.
The AtPQL family can be divided into three groups based on similarity with their non-plant homologues. Detailed sequence analysis was carried out to investigate conserved motifs, topology, domain organisation and genome distribution of the six genes. The implications of these findings for putative functions are discussed.
AtPQL gene expression was investigated using promoter::GUS fusions and quantitative PCR. Whereas AtPQL4 and AtPQL6 appear to be expressed ubiquitously, AtPQL1, AtPQL2 and AtPQL5 are all preferentially expressed in floral tissues and AtPQL3 is expressed primarily in roots. Several family members showed rhythmic expression over the course of a day and induction by both viral infection and wounding. Localisation at the sub-cellular level was investigated using green fluorescent protein fusions and confocal microscopy. At least four AtPQL proteins are targeted to intracellular compartments. AtPQL1 and AtPQL5-GFP fusions both highlight the tonoplast whereas AtPQL4 and AtPQL6-GFP appear to highlight the ER.
A number of knockout and over-expresser mutants were isolated for each gene. Although no physiological phenotype could be found, mutants with altered expression of AtPQL4 and AtPQL6, the two closest A. thaliana homologues of MPDU1/LEC35, showed a clear molecular phenotype. These mutants displayed differential regulation of a number of genes encoding N-glycoproteins as well as defence, cell-surface, lipid biosynthesis and ER stress associated proteins.
Finally, the functional importance of the AtPQL family is discussed and a role for AtPQL4 and AtPQL6 in glycosylation proposed.
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