Mechanisms driving membrane protein targeting and insertion in a synthetic minimal cell

Eaglesfield, Ross (2020) Mechanisms driving membrane protein targeting and insertion in a synthetic minimal cell. PhD thesis, University of Glasgow.

Abstract

The generation of an amphiphilic, semi-permeable membrane was one of the key events in the evolution of cellular life. This event must have been linked to the emergence of integral membrane proteins providing cells with the capability to control the exchange of materials with the environment. Modern cells have evolved complex mechanisms to recruit, insert and assemble these highly hydrophobic proteins into the membrane. Given that many of the proteins involved with these mechanisms are themselves membrane-integral it seems likely that early in evolutionary history cells used more fundamental processes for the insertion of membrane proteins. Identifying such processes is essential for building up an understanding of the more complex mechanisms operating in modern cells and for building artificial cells, both of which will contribute to the advance of medicine and biotechnology.

This thesis has examined the fundamental characteristics responsible for the targeting and insertion of two polytopic α-helical membrane proteins, proteorhodopsin and galactose permease, in a minimal cell-mimicking system. A recombinant cell-free protein synthesis system was used in conjunction with giant unilamellar lipid vesicles (GUVs) to analyse the innate membrane targeting and insertion capability of both proteins in the absence of other targeting and insertion pathways. The results obtained show that both proteins are able to localise and insert co-translationally into the vesicle membrane despite the large aqueous volume of the GUV lumen. Removal of the N-terminal hydrophobic regions of both proteins led to mislocalisation and increased aqueous aggregation, highlighting the importance of effective recruitment of translating ribosomes to the membrane and of the N-terminal regions in this process, even in the absence of other chaperoning mechanisms. Follow-up experiments using truncated proteins, as well as stalled and bound ribosomes, unravelled the functions of the N-terminal domains in ribosome recruitment and insertion.

Taken together, the data obtained supports a model in which the first hydrophobic portion of the protein to emerge from the ribosome effectively recruits the translating ribosome nascent chain complex to the membrane, followed by spontaneous membrane insertion of the entire protein. This protein inherent mechanism represents a simple, fundamental mode of membrane protein biogenesis, which is likely to be masked by the existence of more sophisticated chaperoning and translocation pathways in the complex subcellular context of modern cells. The technology developed in this thesis offers new opportunities to de-construct in vivo pathways and to build artificial cells from the bottom up.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Amtmann, Prof. Anna and Reboud, Dr. Julien
• Date of Award: 2020
• Depositing User: Mrs Marie Cairney
• Unique ID: glathesis:2020-78979
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Jan 2020 16:11
• Last Modified: 30 Aug 2023 08:33
• Thesis DOI: 10.5525/gla.thesis.78979
• URI: https://theses.gla.ac.uk/id/eprint/78979