Transport of glutathione across the endoplasmic reticulum membrane

Szmaja, Tomasz (2018) Transport of glutathione across the endoplasmic reticulum membrane. PhD thesis, University of Glasgow.

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γ-L-glutamyl-L-cysteinylglycine known as glutathione is tripeptide synthesised in the cytoplasm. Glutathione plays important role in several cellular processes and has many functions, for example detoxification of xenobiotics, oxidative protein folding, protection against reactive oxygen species, modulation of cell proliferation and apoptosis. Glutathione is present in almost all organelles within the cell, however, it is still not known how glutathione is transported from the cytoplasm to other cellular compartments. Here we investigated the transport of glutathione into the ER where glutathione plays an important role in oxidative protein folding. Two assays to monitor glutathione transport across the ER membrane were developer and an attempt to identify the putative glutathione transporter was made. Both assays rely on selective permeability of biological membranes and use microsomes to mimic the ER environment. Microsomes were prepared from HT1080 cells expressing either redox sensitive green fluorescent protein (roGFP-iE) or glutathione S-transferase P1 (GSTP1-1A) inside the ER. By measuring the change in fluorescence of roGFP-iE it was possible to measure the rate at which glutathione is transported inside microsomes. GSTP1-1A is able to conjugate glutathione to various substrates and form a stable product, by measuring the increase in glutathione conjugates it was possible to estimate the transport of glutathione inside microsomes. Using both roGFP-iE and GSTP1-1A based assays we were able to measure glutathione transport into microsomes, both assays provide slightly different information about glutathione transport and both have different limitations. In order to identify the glutathione transporter, we used GSH as an affinity ligand and isolated all ER membrane proteins interacting with glutathione. The first approach using glutathione Sepharose beads allowed to isolate several proteins, however, all of them belonged to GST family. A second approach relied on isolating the transporter using glutathione attached to Mts-Aft-Biotin a photo activated crosslinker. Mts-Aft-Biotin approach did not result in isolation of any proteins binding specifically to GSH, this approach requires more work and needs to be improved. We showed that it is possible to isolate GSH binding proteins using GSH as affinity ligand, using this strategy it might be possible to isolate GSH transporter in the future. Both assays presented in this work are the first assays specific for the transport of glutathione across the ER membrane. In the future these assays can be used to investigate the transport of glutathione even further and contribute to the understanding of redox homeostasis in the ER.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Glutathione, GSH, CDNB, transport, GST, BODIPY, redox, ER, Endoplasmic reticulum.
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology > Molecular Cell and Systems Biology
Supervisor's Name: Bulleid, Professor Neil and Hartley, Professor Richard
Date of Award: 2018
Depositing User: Doctor Tomasz Szmaja
Unique ID: glathesis:2018-9112
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 22 May 2018 11:05
Last Modified: 01 Nov 2018 10:45

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