Thesis of Marine Houdou

Amphithéâtre Pierre Glorieux (CERLA) Thesis defense of Marine Houdou - laboratory UGSF-CCHB PhD supervisor : François Foulquier Regulation of cellular Mn homeostasis : unexpected functions of TMEM165, SERCA and SPCA1 Abstract : Glycosylation is a universal cellular process in all living organisms where monosaccharides are added one by one onto an acceptor molecule, most of the time a protein, a lipid or another monosaccharide. In eukaryotes, many glycosylation pathways occur simultaneously, resulting in the biosynthesis of a broad variety of glycan structures with different functions. In humans, if one -or more- glycosylation reactions are genetically impaired, Congenital Disorders of Glycosylation (CDG) appear. One of them, TMEM165-CDG, was identified in 2012 by our group and is at the heart of this work. Pathogenic mutations in TMEM165 gene cause severe glycosylation defects mainly characterized by hypo-galactosylated N-glycan structures. While characterizing these glycosylation abnormalities, a link has rapidly been established by the team between TMEM165 deficiency and Golgi manganese (Mn²⁺) homeostasis disruption. Therefore, and based on previous work, TMEM165 was assumed to act as a Ca²⁺/Mn²⁺ antiporter, allowing the import of Mn²⁺ into the Golgi lumen in order to sustain an adequate ionic environment, required for all glycosylation reactions. Interestingly, we also found that exogenous addition of Mn²⁺ in the culture medium of TMEM165 deficient cells completely rescues the N-glycosylation defects observed in these cells. Moreover, TMEM165, like Gdt1p its yeast ortholog, is a protein highly sensitive to Mn²⁺, being rapidly degraded via the lysosomal pathway in the presence of high Mn²⁺ concentrations. All in all, a close link exists between TMEM165/Gdt1p, Golgi Mn²⁺ homeostasis and Golgi glycosylation; the three major aspects focused in the PhD research. More precisely, my thesis focuses on (i) understanding the mechanisms of Mn²⁺-induced glycosylation rescue in TMEM165 deficient cells and (ii) the potential links between different key players acting in the regulation of the secretory pathway ionic homeostasis which are the Sarco/Endoplasmic Reticulum calcium (Ca²⁺)-ATPase SERCA2, TMEM165 and SPCA1 (Secretory Pathway Ca²⁺/Mn²⁺-ATPase), the only pump of the Golgi apparatus known to import both Ca²⁺ and Mn²⁺ in the Golgi lumen. Through the use of isogenic human cell lines knockout for either TMEM165 or SPCA1 and yeasts lacking Gdt1p and/Pmr1p, we highlighted three main concepts that closely link these proteins: TMEM165 (Gdt1p), SPCA1 (Pmr1p) and SERCA2. On the one hand, we demonstrated that the activity of SERCA pumps is crucial to sustain Golgi glycosylation reactions in absence of TMEM165 by their contribution in Mn²⁺ pumping and redistribution into the Golgi lumen. On the other hand, TMEM165 was found essential for maintaining Golgi glycosylation reactions in absence ofSPCA1 and when SERCA2 are inhibited by pharmacological agents. Moreover, we also shed light on the fact that expression and stability of TMEM165 (in humans) and Gdt1p (in yeast) were directly linked to the capacities of SPCA1 and Pmr1p to import Mn²⁺ into the Golgi lumen. Although differences exist between humans and yeast Saccharomyces cerevisiae, all of our work illustrates the crucial importance of the ionic homeostasis of the Golgi apparatus to sustain Golgi glycosylation reactions.