SOTTODOMINI DEL RETICOLO ENDOPLASMATICO IN FISIOLOGIA E PATOLOGIA

The endoplasmic reticulum, the largest organelle of eukariotic cells, presents a very complex architectonical organization that reflects its several functions. It is now well known how this organelle presents many sub-domains to exploit particular functions, such as transport throughout the secretory pathway and degradation of misfolded proteins; sub-domains in the ER can be created also after pathological events. My Thesis project is dedicated to characterize two sub-domains of the endoplasmic reticulum, one correlated to pathology, and another one commonly present in healthy cells. Regarding the sub-domain generated in pathological context, I studied VAPB, a membrane protein with a C-terminal anchor (“tail-anchored”), which is resident in the endoplasmic reticulum, whose P56S mutation induces large perinuclear inclusions formation. Considering that such aggregates haven’t still clearly related to any cellular organelle, I decided to study the biogenesis of the wild-type and mutated protein both in vivo and in vitro, and characterize mutant protein generated aggregates. In vitro assays are based on the N-glycosilation of an epitope anchored to the C-terminus of the protein and also on protease protection assays developed in my laboratory, while in vivo experiments are aimed to the morphological evaluation of the inclusions induced by the mutant protein. With such approaches we have demonstrated that both the wild type form and the mutant one insert the membrane of the endoplasmic reticulum, following the post-translational pathway, and the in vitro behaviour of the mutant is indistinguishable from the one of the wild type. However, in vivo, at very early times from the insertion into the endoplasmic reticulum (2 hours), P56S-VAPB organizes into small clusters that then fuse to form large paranuclear inclusions. Such inclusions are positive for some endoplasmic reticulum markers, while they result negative for markers of other organelles. The inclusion continuity with the surrounding endoplasmic reticulum has been demonstrated with FRAP and FLIP analysis. The ultrastructural analysis demonstrated that the inclusions consist of pair of cisternae intermingled by a cytosolic layer of approximately 30 nm. This form of OSER is probably generated by high affinity trans interactions between the cytosolic domains of P56S-VAPB. These results demonstrate that P56S-VAPB causes a dramatic remodelling of the endoplasmic reticulum that can explain the neurotoxic action of the protein. Regarding the role of endoplasmic reticulum sub-domains in physiology, I focused on a possible role of lipid microdomains in the segregation and plasma membrane transport of membrane proteins. As models for my study I used an endoplasmic reticulum resident protein, cytochrome b5, and an its variant, b5-22, different from the wild-type protein only for its transmembrane domain extended of only 5 aminoacids. Previous studies in my laboratory demonstrated that the b5-22 variant inserts initially in the endoplasmic reticulum, but then it distributes disomogenously within the organelle, concentrating into the tubules and into the exit sites. To investigate the possible role of lipid microdomains in such event, I developed an experimental approach based on the use of radiolabelled, photoactivable lipid probes and I so analyzed the lipid environment of the endoplasmic reticulum in the proximity of b5 wild type and b5-22. With such approach I observed that b5-22 is less accessible to phosphatidylcholine in comparison to the wild-type. This observation suggests that b5-22 distributes into microdomains that are poor of phosphatidylcholine and so enriched of another lipid. Such segregation could lead to the concentration of b5-22 into the exit sites and so the its export from the endoplasmic reticulum. The results obtained illustrate how protein-lipid interactions based on simple physical-chemical parameters can determine the fate of a protein throughout the secretoty pathway.