Spatiotemporal dynamics of COPII vesicle formation and morphogenesis of exit sites in the endoplasmic reticulum
Zusammenfassung der Projektergebnisse
The central focus of the project proposal was on the spatiotemporal organization of exit sites in the endoplasmic recticulum (ERES) and on the regulation of COPII proteins at ERES. To gain insights into the different facets of these events, it was proposed to monitor fluorescently tagged proteins and/or lipids by advanced light microscopy techniques (e.g. fluorescence correlation spectroscopy and fluorescence recovery after photobleaching) on dedicated giant unilamellar vesicles (GUVs) and in transiently/stably transfected cell lines. Based on the experimental data, quantitative models for the spatiotemporal dynamics of COPII proteins at ERES and the self-organization of ERES as individual entities were envisaged. As a result of our experimental efforts, we have found that neither GUVs made from artificial lipid mixtures that mimic ER membranes nor GUVs made from native ER material show a spontaneous phase separation into domains. These results are in agreement with observations of other research groups who did not even observe domain formation upon addition of peripheral membrane proteins, e.g. GTPases like ARF-1 or Sar1. These data strongly indicate that lipids are not the major organizers of membrane domains in the ER but ERES seem to emerge due to the interaction of proteins. Along these lines, we have started a collaboration with Hesso Farhan (Biotech. Inst. Thurgau, Kreuzlingen, CH) in the late phase of the project period to elucidate the regulating role of the peripheral membrane protein Sec16 in the formation of ERES. Preliminary results indicate that ERES number and size are regulated by Sec16 in response to changes in the status of growth factors. Also, Sec16 seems to change its oligomeric state upon phosphorylation and as a consequence of interactions with COPII proteins. Work along these lines will be followed up also after the funding period. As a result of our modeling and simulation work, we have been able to show that protein acylation (e.g. palmitoylation) in conjunction with the proteins’ hydrophobic mismatching can serve as a basic means of sorting and segregation of transmembrane proteins. Protein sorting at the level of the ER (and beyond) therefore may rely on this robust physicochemical strategy that can be further fine-tuned by well-known biochemical motifs, e.g. a cytoplasmic di-arginine. We also found that peripheral membrane proteins like the small GTPase Sar1 specifically deform lipid bilayers. The associated lipid perturbations can lead to attractive membrane-mediated interactions that drive protein oligomerization within one leaflet but also across leaflets. Hence, this mechanism may support the formation of nucleation seeds for COPII vesicles and/or ERES. In an ongoing project in collaboration with the Pepperkok group (EMBL) we found that p150glued, a part of the dynein-associated dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. As a consequence, concentration of cargo is reduced in the presence of an antagonistically acting C-terminal fragment of p150glued that prevents binding of endogenous p150glued to Sec23p. This suggests that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules.
Projektbezogene Publikationen (Auswahl)
- On the role of acylation of transmembrane proteins. Biophys. J. 98, 800 (2010)
D. Morozova & M. Weiss
- Dynamic structure formation of peripheral membrane proteins. PLOS Comp. Biol. 7, e1002067 (2011)
D. Morozova, G. Guigas & M. Weiss
- Membrane-mediated interactions – a physico-chemical basis for protein sorting. Mol. Membr. Biol. 29, 177 (2012)
M. Hanulova & M. Weiss
(Siehe online unter https://doi.org/10.3109/09687688.2012.667838) - Protein sorting and membrane-mediated interactions. Biophys. Rev. 4, 117 (2012)
M. Hanulova & M. Weiss
