2026-09-25T09:52:29Zhttps://mro.massey.ac.nz/server/oai/requestoai:mro.massey.ac.nz:10179/72972025-11-12T14:04:42Zcom_10179_294col_10179_296
DSpace at Massey University
author
Shanmugam, Renuka
2015-10-29T21:53:26Z
2015-10-29T21:53:26Z
2015
http://hdl.handle.net/10179/7297
Q112910635
https://www.wikidata.org/wiki/Q112910635
All cells must have the ability to deal with a variety of environmental stresses.
Failure to adapt and protect against adverse stress conditions can lead to cell
death. One important stress that affects all cells is amino acid limitation. Amino
acids are building blocks of proteins. Gcn2 is a protein kinase, activated under
conditions of amino acid limitation and the active Gcn2 reduces the general
protein synthesis and specifically increases the synthesis of a protein called Gcn4,
a transcription factor of stress response genes.
Gcn2 is found in virtually all eukaryotes. In addition to the amino acid limitation
it protects cells to a large array of stress conditions such as glucose and purine
limitation, high salt, reactive oxygen species and UV irradiation. Interestingly,
Gcn2 has been found to have acquired additional functions in higher eukaryotes
such as cell cycle regulation, viral defense and memory formation. Not
surprisingly, Gcn2 has been implicated in diseases and disorders such as abnormal
feeding behaviour, cancer, Alzheimer’s disease, impaired immune response,
congestive heart failure, and susceptibility to viruses including HIV. Despite of its
medical relevance, so far it is unknown how the cell ensures proper Gcn2
function.
Yeast studies have uncovered that for almost all Gcn2 functions Gcn2 must bind
to its positive effector protein Gcn1. Gcn1 is proposed to be a scaffold protein,
strongly suggesting that it serves as a platform for recruiting other proteins close
to Gcn2 to fine-tune its activity. For this reason, in this study, we set out to
comprehensively identify all proteins binding to Gcn1, i.e. generate the Gcn1
interactome, using a procedure that allowed us to also identify proteins that only
weakly or transiently contact Gcn1 (a typical property of regulatory proteins). We
have identified several potential Gcn1 binding proteins from published and in
house data. Sixty six of these were further analyzed using the respective deletion
strains. Ten of these deletion strains were unable to grow under amino acid
starvation conditions. Five of these showed reduced eIF2! phosphorylation,
strongly suggesting that they are positive effectors of Gcn2. Using plasmids from
the Yeast Genome Tiling Collection, we were able to rescue the Gcn2 function of
three deletion strains (kem1", msn5" and sin3"), indicating that the defect was
due to the deletion of the respective gene. In addition, some of these proteins were
confirmed to reciprocally bind to Gcn1. Finally, we show that Kem1 partially
facilitates activation of Gcn2 via Gcn1 and it may play a role as a positive
regulator of Gcn2. Furhther the interactions were validated by reciprocal
immunoprecipitation. Taken together, this study sheds light on novel Gcn1
binding proteins regulating Gcn2.
en
The Author
Proteins
Amino acids
Gcn1
Gcn2
Amino acid limitation
Cellular stress
Identification of Gcn1 binding proteins and characterization of their effect on Gcn2 function : a thesis submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biochemistry, Massey University, Albany, New Zealand
Thesis
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