ANALYSIS OF THE YEAST PLASMA MEMBRANE IN Saccharomyces cerevisiae USING SUPER RESOLUTION MICROSCOPY

The plasma membrane (PM) separates the cell from its environment. In budding yeast S. cerevisiae, PM exhibits prominent patterns of membrane compartmentalization and three such microcompartments have been described,namely MCC (microcompartment of Can1), MCP (microcompartment...

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Bibliographic Details
Main Author: MEUTIAWATI (NIM : 20511013); Pembimbing 1 : Dessy Natalia, PhD; Pembimbing 2 : Prof. Ber, FEBRINA
Format: Theses
Language:Indonesia
Online Access:https://digilib.itb.ac.id/gdl/view/15656
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Institution: Institut Teknologi Bandung
Language: Indonesia
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Summary:The plasma membrane (PM) separates the cell from its environment. In budding yeast S. cerevisiae, PM exhibits prominent patterns of membrane compartmentalization and three such microcompartments have been described,namely MCC (microcompartment of Can1), MCP (microcompartment <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> of Pma1) and MCT (microcompartment of target of rapamycin kinase 2). Theappearances of these domains under the microscope are ranging from discrete patches (MCC, MCT) to homogenous networks (MCP). Studies about the organization of the yeast PM have been done mostly using fluorescence and electron microscopy. The aims of this study were to develop tools for determining the localization of individual proteins in the PM and to compare the imaging of <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> plasma membrane proteins between confocal and super resolution microscopy analysis (photoactivated light microscopy, PALM). It has been reported that the C-terminus sequences derived from three amino acid transporters, namely Gap1, Bap2 and Hip1 allowed reporter proteins to associate with the PM (Bert Poolman, <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> unpublished data). All three sequences contained the FWC (Phe-Trp-Cys) signature which could be used to generate tools for PM architectural analysis. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Eight of amino acid transporters in the yeast PM, namely Tat1, Tat2, Sam3, Agp1, and Gnp1 contain FWC signatures while one of them contains FFC signature in the C-terminus. Green fluorescent protein (GFP) was fused to both the full length and the C-terminus segments of these proteins. GFP tagged C-terminus constructs of the amino acid transporters showed plasma membrane localization under the confocal microscope. Addition of FWC to the C-terminus of the other PM proteins associated these segments with the PM, while it had no effect on the <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> localization of C-terminus segments derived from vacuolar membrane proteins. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> The C-terminus segments containing FWC were less prone to aggregation and gave better PM staining than their corresponding full length proteins. Thus, these C-terminus segments were used as a tool for the PALM analysis. Gene constructs containing PSmOrange, mEos3.1 and mEos3.2, photoswitchable fluorescent proteins that are suitable for PALM, were generated. Gap1 C-terminus and Can1 full length showed a nuclear staining and no photoactivation in PALM. <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> Meanwhile, confocal microscopy imaging of those constructs showed no expression of the fluorescent protein PSmOrange after induction with 0.2% galactose for 2 hours. Using different photoswitchable proteins, Meos3.1 and mEos3.2-Gap1C-terminus showed homogenous distribution in the PM, while mEos3.1 and mEos3.2-Hip1C-terminus appeared to be discrete patches in the PM. Single molecule localization was achieved with PALM and the resolution of the image reconstructed with PALM was found to be 5 times higher than the image captured with confocal microscopy.