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Targeting of a calcium-sensitive fluorescent probe into the Golgi apparatus of the yeast Saccharomyces cerevisiae

Vanden Hoek, Elina
(2022)

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Details

Supervisors
Morsomme, Pierre ; Lejeune, Quentin
Faculty
Faculté des bioingénieurs
Degree label
Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée
Abstract
Gdt1p is a transporter localized in the Golgi apparatus of the yeast Saccharomyces cerevisiae involved in calcium, manganese and pH homeostasis. This protein would act as a cation exchanger transporting calcium and manganese against protons or calcium against manganese. However, studies are still to be carried out to fully characterize the mechanism of Gdt1p such as the direction of cation transport. Moreover, the concentration of calcium in the Golgi apparatus is not precisely known. To improve our knowledge about Gdt1p and its role in calcium homeostasis, in vivo measurements of Ca2+ concentration in Golgi apparatus are required. Therefore, several genetically encoded calcium indicators (GECI) were engineered to target this organelle. Unfortunately, these previous attempts were unsuccessful. The tested sensors were indeed not sensitive to calcium once expressed in Saccharomyces cerevisiae. The aim of this project is to engineer a calcium-sensitive probe suitable for in vivo measurements in Golgi apparatus, the FRET-based D4cpV sensor. This sensor is composed of the calmodulin calcium binding site and M13 peptide cloned between ECFP and circularly permuted Venus (two variants of the fluorescent protein GFP). With its dissociation constant about 64 μM, the D4cpV probe would be adapted to the Golgi apparatus in which the calcium concentration would be around 10 μM to 300 μM. To target the D4cpV probe into the Golgi apparatus (GoD4cpV), its N-terminus was fused to the transmembrane domain of Mnn2 (an intrinsic Golgi protein) through a 3Gly-HA-3Gly linker. In this master thesis different steps are presented, from the genetic construction to the characterization of the sensor. Results led to the conclusion that the probe was well expressed even if some degradation products were detected by Western Blot. Normally localized into the Golgi apparatus due to Mnn2p, the GoD4cpV was also localized in the endoplasmic reticulum. By permeabilizing cells with 0.16% digitonin, a detergent, we were able to confirm the calcium sensitivity of the probe. Nevertheless, improvements are still needed to correctly calibrate the probe.