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Characterisation of a newly identified family of lipid transfer proteins at membrane contact sites

Gatta, A; (2016) Characterisation of a newly identified family of lipid transfer proteins at membrane contact sites. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Non-vesicular intracellular lipid traffic is mediated by lipid transfer proteins (LTPs), which contain domains with an internal cavity that can solubilise and transfer lipids. One of the most widespread LTP folds is the Steroidogenic Acute Regulatory Transfer (StART) domain, which forms a hydrophobic pocket, and appears in proteins with different localisations and lipid specificities. The aim of this study was to characterise a new StART-like domain family, which we identified by a bioinformatics approach. I studied aspects of the localisations, functions and structural properties of six StART-like proteins in S. cerevisiae. The yeast StART-like proteins were endoplasmic reticulum (ER)-integral membrane proteins with transmembrane domains, and they localised at membrane contact sites: Lam1p/Lam3p, and Lam2p/Lam4p at junctions between ER and plasma membrane (PM); Lam5p/Lam6p at junctions between the ER and the vacuolar membrane, at nucleus-vacuole junction (NVJ) and at ER-mitochondria contacts. To study their functions, I purified the second StART-like domain of Lam4p, and I identified sterol as its lipid ligand from in vitro binding assays and in a spectroscopy approach with fluorescent ergosterol. We named the whole family LAM for Lipid transfer proteins Anchored at Membrane contact sites. The sterol binding property of the domains was related to a phenotype shared by LAM1, LAM2 and LAM3 delete strains, which showed an increased sensitivity to the sterol-sequestering polyene antifungal drug Amphotericin B (AmB). The two most sensitive strains (lam1∆ and lam3∆), displayed low sphingolipid levels, which is as yet unexplained. All AmB phenotypes were rescued by StART-like domains from the human LAMa, Lam2/4p and Lam5/6p, suggesting that these domains bind sterol. Simultaneous deletion of LAM1, LAM2, and LAM3 significantly reduced the extent of cortical ER-PM contacts, implying that they create the structure of the particularly punctate contact site they target. Finally, I started structural analysis of Lam4S2 to study the mechanism of sterol binding and to confirm our structural model.

Type: Thesis (Doctoral)
Title: Characterisation of a newly identified family of lipid transfer proteins at membrane contact sites
Event: University College London
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Third party copyright material has been removed from ethesis.
Keywords: Cell biology, Lipid homeostasis, Membrane trafficking, Lipid transfer protein, Membrane contact sites, Saccharomyces cerevisiae, Structural biology, Biochemistry, Bioinformatics, Cholesterol homeostasis
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
URI: https://discovery.ucl.ac.uk/id/eprint/1517331
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