Study of the role of the membrane protein Smf2p on manganese homeostasis in Saccharomyces cerevisiae

Details

Supervisors

Morsomme, Pierre

Faculty

Faculté des bioingénieurs

Degree label

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

Abstract

enSmf2p is a yeast transporter from the Nramp family believed to be localized in Golgi-like vesicles and late endosomes. The role of this protein in manganese transport is not fully understood yet, but a growing consensus supports that it transports it from the secretory pathway to the cytosol when needed elsewhere within the cell. However, these hypotheses regarding its localization and its role on manganese homeostasis still require direct experimental validation. The aim of this project is to bring additional information on the role of Smf2p on manganese homeostasis in Saccharomyces cerevisiae by assessing the manganese content within the cell and some of its constituent when different manganese transporters are deleted. In parallel, additional information on the possibility of manganese export via the secretory pathway can be illustrated by those same measurements with the use of the thermosensitive sec1-1 mutation, preventing exocytosis from happening. Two types of experiments can be conducted to study the manganese concentration in the cells. A first one using inductively coupled plasma - atomic emission spectroscopy (ICP-AES) can be used to assess the total manganese levels within cells. And a second one using manganese-sensitive fluorescent probes, based on the FRET mechanism, can be used to measure the in vivo relative manganese content in the cytosol and in the Golgi of yeast cells. Those sensors, MnLaMP1 and 2, have just recently been engineered with the use of the lanmodulin protein as a manganese binder between two GFP variants: ECFP (donor fluorophore) and cpV (acceptor fluorophore). In this master thesis, cytosolic probes were genetically constructed using the previously constructed Golgi-targeted probes as templates, removing their Mnn2 transmembrane domain and HA-tag by PCR, and re-cloning the resulting MnLaMP sequences into an empty pRS315 backbone for cytosolic expression. They were then genomically integrated in the his3∆1 locus of yeast strains. The combination of total cellular manganese quantification by ICP-AES and complementary fluorescence measurements proved that manganese can be exported form the cell via exocytosis in certain conditions. Notably, this export does not occur when the Pmr1p transporter is absent. While the involvement of Smf2p could not be conclusively demonstrated with those results, incorporating vacuole-related measurements in future work holds promising potential to clarify its function.