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Title: Functional characterisation of a predicted chloroplastic plant protein phosphatase
Author: Seymour, Michael David John
ISNI:       0000 0001 3394 8576
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 2009
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The phosphatase AtPTPKISI is involved in the control of starch metabolism in Arabidopsis thaliana leaves at night. The SEX4 (Starch Excess 4) mutants, lacking this predicted phosphatase, have strongly reduced rates of starch metabolism. It is shown that this chloroplastic protein can bind to glucans through a carbohydrate binding domain (CBM) located within its previously predicted kinase interaction sequence (KIS), while another novel KIS containing protein (AKINβy) shows no such interaction. Further analysis of the CBM identifies conserved residues vital for carbohydrate binding and common to CBMs, as well as sugar tongs, not present in similar CBMs or the GBD/KIS domain of the previously studies AMPKβ, but found within the binding domain of the PTPKIS family proteins. While PTPKIS 1 shows activity to generic phosphatase substrates, it is unable to dephosphorylate either phosphotyrosine or phosphothreonine containing peptides. It does however show phosphatase activity towards phosphorylated starch and amylopectin, comparable to that of the mammalian protein laforin. Remarkably, the most closely related protein to PTPKIS 1 outside the plant kingdom is laforin, required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans, through the formation of insoluble starch like polyglucans (lafora bodies). In addition to PTPKIS 1, PTPKIS 2 (At3g015180) is identified, a predicted phosphatase, with a domain structure homologous to that of PTPKIS 1, termed. The PTPKIS2-SALK (PTPKIS2 knockout) mutant, lacking this predicted phosphatase, has a reduced rate of starch metabolism. It is shown that this mutant causes a phenotype similar to SEX4, but less extreme. It is further shown that this protein can bind to glucans through a carbohydrate binding domain (CBM), but unlike PTPKIS 1 shows no activity towards any phosphate substrates. PTPKIS2 does however modulate the activity of PTPKIS 1, causing a 4-fold increase in the activity of PTPKIS 1 against phosphorylated starch, when both enzymes are present in equimolar concentrations. Finally, a hypothesis is proposed as to the roles of PTPKIS 1 and PTPKIS 2 in starch metabolism, and the similarity of function seen in the mammalian protein Laforin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available