Engineering of a fluorescent probe for in vivo measurements of Ca2+ concentrations in the yeast Golgi apparatus

Details

Supervisors

Morsomme, Pierre ; Lejeune, Quentin

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Gdt1 is a yeast cation transporter localized in the Golgi apparatus membrane. This protein is involved in calcium, manganese, and pH homeostasis. To date, two hypotheses exist concerning its transport mechanism. Whilst the first states that Gdt1 transports either calcium or manganese ions against protons, the second supports that calcium ions are exchanged against manganese ions. However, additional studies are still needed to fully characterize the mechanism of this transport. More globally, the understanding of Gdt1 in yeast model brings additional information about its human ortholog TMEM165, which constitutes a larger scale objective. Indeed, mutations in the genetic sequence of TMEM165 lead to specific forms of congenital disorder of glycosylation (CDG), resulting from a failure in protein N-glycosylation process. Additional information can be brought by conducting in vivo measurements of calcium concentration in the Golgi apparatus ([Ca2+]Golgi). These experiments can be carried out using genetically encoded calcium-sensitive indicators (GECI) engineered to target this organelle. Many probes of this type have already been tested in our lab without concluding results. However, a probe named D4cpV has been successfully engineered recently by Quentin Lejeune and Elina Vanden Hoek. This sensor is based on the FRET mechanism and is composed of two GFP variants: an ECFP (donor fluorophore) and a cpVenus (acceptor fluorophore). Finally, this probe is targeted into the Golgi membrane using the sequence of the transmembrane domain of Mnn2, an intrinsic Golgi protein. This work focuses on the calibration of this probe in yeast. Firstly, Golgi vesicles purification allowed to confirm the sensitivity of the probe to calcium using a fluorimeter. Then, in vivo [Ca2+]Golgi was measured following an extracellular stress to determine whether calcium transport could be detected across the Golgi apparatus membrane. At first sight, no proof of such transport can be observed following this stress. However, the sensor’s sensitivity is finally questioned.