The team in 2019 (from left to right): Guillemette van Raemdonck, Camille Lemaigre, Benjamin Ledoux, Sylvain Nootens, Louise Thines, Henri-François Renard, Antoine Deschamps, Jiry Stribny, François Tyckaert, Pierre Morsomme
(Missing on the picture: Alexandra Gaiffe, Quentin Lejeune and Shiqiang Xu).
Our lab is studying the function, biochemistry, regulation, structure and trafficking of membrane proteins with a particular focus on membrane transporters.
At the origin, we focused on the heterologous expression, purification, reconstitution and characterization of plant and archea plasma membrane H+-ATPase genes in the yeast Saccharomyces cerevisiae. Then, we moved on to the fascinating topic of membrane trafficking that is how membrane proteins are transported within the cell to reach their final destination.
First, we studied the transport of membrane proteins through the secretory pathway, especially from the ER to the Golgi apparatus. Then we studied the trafficking of small membrane proteins, Sna1p, Sna2p, Sna3p, and Sna4p, which all belong to the same family, but are located in different subcellular compartments. We identified specific sequences responsible for the targeting of Sna proteins to their final destination: the PPxY motif for vacuolar degradation pathway, the ExxLL motif for the vacuolar membrane, and the tyrosine motif (Yxxf) for ER exit and vacuolar membrane.
Besides we have developed an optimized plasma membrane purification procedure and a quantitative gel-free proteomic approach to monitor dynamic changes in the plasma membrane proteome. This was developed thanks to the Massprot platform. This procedure has been first developed in yeast and led to the identification of about 120 integral plasma membrane proteins representing 50% of the plasma membrane proteome. We measured the changes which appeared when cells were submitted to external stresses (salt stress, nutrient availability changes…). In particular, we have characterized the endocytic mechanism by which the thiamin transporter Thi7p is controlled by the presence or not of thiamin in the external medium.
Then, with Henri-François Renard (now professor at UNamur), we have adapted our proteomic approach to study new endocytic pathways in mammalian model systems. Our idea was to perform a quantitative proteomic screening of human cell surface proteins in conditions where canonical clathrin-dependent endocytosis is blocked. Our hypothesis was that, in these conditions, clathrin-dependent cargoes should accumulate at cell surface, while others should not change. Indeed, we observed a strong and specific accumulation of canonic clathrin cargoes such as transferrin and LDL receptors at the plasma membrane. However, in the same conditions, we noticed that several cargoes were depleted from the plasma membrane. The cell surface immunoglobulin-like glycoprotein CD166/ALCAM was the most extreme case of cell surface down regulation, suggesting that this protein might still be endocytosed and degraded. This exciting finding led us to hypothesize that CD166 is a new clathrin-independent cargo. We characterized this new endocytic pathway and found that the cytosolic BAR domain protein Endophilin A3 specifically and functionally associates with CD166-containing early endocytic carriers. In addition, we found that the construction of endocytic sites from which CD166 is taken up in an endoA3-dependent manner is driven by extracellular galectin-8. CD166 protein is heavily glycosylated and it is a binding partner of galectin-8. Interestingly, galectins have been proposed to cluster glycosylated cell surface cargo proteins and glycosphingolipids to promote endocytosis of these cargoes. This new EndoA3/Gal8-dependent mechanism of endocytosis is essential to control the abundance of CD166.
Finally, we also discovered that a modification of the abundance of CD166 at the cell surface modifies the adhesive and migratory properties of cancer cells.
Figure 1: Working model for endoA3-mediated endocytosis of CD166 (from Renard et al., 2020). In the meantime, a new research project has been developed towards the study of uncharacterized membrane transporters some of them being involved in genetic diseases in human.
In parallel, our group has participated to the discovery of a new family of cation transporters linked to genetic diseases, the Congenital Disorders of Glycosylation (CDG), which are a heterogeneous group of inborn errors directly or indirectly affecting the glycosylation pathway. In 2012, mutations in a newly identified human gene have been identified as a cause of CDG. This gene, called TMEM165 in humans, is highly conserved throughout evolution and orthologs can be found in many bacteria and all eukaryotes, including the yeast Saccharomyces cerevisiae. It belongs to the UPF0016/GDT1-like family. Bioinformatic analysis reveals that the majority of its members have 6 transmembrane domains (TMDs), contain two copies of a highly conserved motif, E-x-G-D-(KR)-(TS) which are predicted to be localized in the first and fourth TMDs and to form a pore for ion transport across the membranes. This in silico analysis suggested that UPF0016/GDT1 family members could be secondary membrane transporters.
Figure 2: Predicted Gdt1p structure and putative hydrophilic pore. The left panel represents a plane projection of the topology, the central panel is the predicted tridimensional structure generated by the AlphaFold algorithm (Uniprot P38301), and the right panel zoom on the putative central pore of the proteins, with highlighted hydrophilic amino acids that could mediate cation transport.
We have shown that the yeast protein from this family, Gdt1p, is localized in the Golgi apparatus and is involved in calcium, manganese and pH homeostasis. Very recently we set up a new assay which allows to measure cations transport by Gdt1p and TMEM165 when expressed in Lactococcus lactis. This assay revealed that Gdt1p is a calcium and manganese transporter. Manganese transport discovery is very promising since it makes a direct link between GDT1/TMEM165 and glycosyltransferases that use Mn2+ as a cofactor. Besides its role in cations transport and the glycosylation pathway, TMEM165 has recently been proposed to play a role in the control of breast cancer cells growth and migration.
From all these data, obtained in yeast, human cells or in heterologous systems, we hypothesize that this new family of membrane transporters are Mn2+- Ca2+/H+ antiporters and that a defect in calcium, manganese and/or pH homeostasis in the Golgi may be responsible for glycosylation defects This hypothesis is currently tested in our lab.
Selected publications from the lab
Demaegd D. et al., (2013) A newly characterized Golgi-localized family of proteins is involved in calcium and pH homeostasis in yeast and human cells, P. Natl. Acad. Sci. USA, 110, 6859-6864.
Thines L. et al., (2018) The Yeast Golgi Gdt1p Is Intimately Related to Manganese Homeostasis Through its Ability to Transport Mn2+ Cations, J. Biol. Chem., 293, 8048-8055.
Savocco J., Nootens S., Afokpa W., Bausart M., Chen X., Villers J., Renard H.F., Prévost M., Wattiez R. and Morsomme P., (2019) The yeast α-arrestin Art2 is the key regulator of the ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters, PLOS Biology, 17, e3000512.
Stribny J. et al., (2020) TMEM165 transports calcium and manganese when expressed in yeast and bacteria, J. Biol. Chem., 295, 3865-3874.
Renard H.F. et al., (2020) Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166, Nature communications, 11, 1457.