Intercellular signalling is essential for multicellular organisms to coordinate growth
and development, and is mediated by a huge variety of proteins. Some signalling
pathways rely on the proteolytic cleavage of membrane proteins by a relatively
newly discovered process of regulated intramembrane proteolysis (RIP), the cleavage
of proteins within a transmembrane domain. There are four classes of
intramembrane cleaving proteases (ICliPs) – Rhomboids, Site-2-proteases, Signal
peptide peptidases and γ-secretase. Of all the ICliPs studied to date, γ-secretase is
unique, as it is comprised of a four-protein complex, and is only found in
multicellular organisms. A vast amount of research is carried out on the γ-secretase
complex, not just because of its role in developmentally important pathways, such as
NOTCH signalling, but also due to its role in Alzheimer’s disease. The β-amyloid
precursor protein (APP) is cleaved by γ-secretase, and defects in this process result in
the release of abnormal peptides that form the senile plaques in the brains of
Alzheimer’s disease patients. Homologues of the four components of γ-secretase
(PRESENILIN (PS), NICASTRIN (NCT), ANTERIOR PHARYNX DEFECTIVE-1
(APH-1) and PRESENILIN ENHANCER-2 (PEN-2)) are found in plants. The aim
of this thesis was to characterise the potential γ-secretase components in Arabidopsis
thaliana, to determine whether they form a complex, and to analyse what role, if any,
they play in plant signalling. The members of the putative Arabidopsis γ-secretase
complex (AtPS1 and 2, AtNCT, AtAPH1 and AtPEN2) were identified through
BLAST searches, and found to be uniformly expressed. Analysis of T-DNA
insertion mutants in each of these genes, and combinations there of, revealed no
gross morphological differences to wild type under normal growth conditions and
when subjected to a range of stresses. Protein fusions to GFP under the control of
the 35S promoter were constructed and stably transformed into plants. AtPEN2:GFP
is expressed throughout the plant, and accumulates in BFA sensitive Golgi bodies in
roots. AtPS1:GFP, only accumulates strongly in developing seeds. Native blue
PAGE was used to look for high molecular weight complexes (HMW) containing
AtPEN2:GFP and AtPS1:GFP. Both fusion proteins were found in similar sized
HMW complexes. A variety of methods were used to look for substrates of the
iv
putative γ-secretase complex in Arabidopsis, and although no specific substrates
were identified, a potential role in seed development has been established