Active modes of the translocon

All proteins are synthesized in the cytoplasm. However some proteins function outside of cells, in the cell membrane, in the endoplasmic reticulum or one of the connected vesicular systems and need to be sorted and accordingly transported. Consequently, the secretory pathway, a highly conserved protein targeting, translocation and membrane insertion system has evolved to cope with this task. The central actor in the secretory pathway is the heterotrimeric Sec61αβγ protein complex also known as the translocon. This protein complex has been shown to act as a transmembrane channel facilitating protein translocation into the lumen of the endoplasmic reticulum, a starting point in protein secretion. The complex also contains a lateral opening, termed ‘lateral gate’ through which proteins have been suggested to insert into the membrane of the endoplasmic reticulum. The mechanics of translocon opening for protein secretion or membrane insertion has been a matter of debate. The results presented here show secondary structure level resolution cryo-electron microscopic structures of the translocon in complex with stalled ribosome-nascent chain complexes carrying substrates with propensity for either secretion or membrane insertion as well as ribosome-bound inactive translocon molecules. The observed differences in secondary structure suggest a model for translocon activity in protein sorting

Structural and functional analysis of the yeast protein Sec13 with an qmphasis on its protein interaction interface

Jezgra omeđena ovojnicom temeljna je osobina eukariota. Kako je kompleks jezgrine pore (NPC) jedini posrednik u transportu između jezgre i citoplazme, rasvjetljavanje njegove strukture važno je za razumijevanje eukariotskih stanica. Sec13 je komponenta NPC čija je uloga u održavanju i stabilizaciji kompleksa nejasna. Njegova dvojna uloga u vezanju proteina Nup145C u sklopu NPC te proteina Sec31 u sklopu vezikula tipa COPII otežava mutacijsku analizu. Cilj ovog projekta bio je riješiti strukturu Sec13 u kompleksu sa insercijskom regijom Nup145C, kako bi se dobila jasna slika veznog mjesta Sec13/Nup145C. Riješena struktura uspoređena je sa strukturama kompleksa Sec13/Sec31. Na temelju razlika među veznim mjestima osmišljen je mutirani Sec13 koji veže isključivo protein Sec31. Posljedica ove mutacije potvrđena je pokusima in vitro.Possession of an envelope-coated nucleus is the defining trait of eukaryotes. Since the nuclear pore complex (NPC) is the only known mediator of nucleo-cytoplasmic traffic, elucidating its structure is important for understanding eukaryotic cells. Sec13 is a component of the NPC whose importance for the stability of the complex is unclear. Its dual role in binding proteins Nup145 in the NPC and Sec31 in COPII vesicles makes mutational analysis of Sec13 function difficult. The goal of this project was solving a structure of Sec13 in complex with the Nup145C insertion blade that would provide a clear view of the Sec13/Nup145C interface. The obtained structure was compared to the structures of the Sec13/Sec31 complex. Based on the differences between the interaction sites of the structures, a mutant Sec13 protein was designed, that binds specifically to Sec31, but has lost affinity for Nup145C. The effect of this mutation was confirmed by in vitro experiments

Structural and functional analysis of the yeast protein Sec13 with an qmphasis on its protein interaction interface

Jezgra omeđena ovojnicom temeljna je osobina eukariota. Kako je kompleks jezgrine pore (NPC) jedini posrednik u transportu između jezgre i citoplazme, rasvjetljavanje njegove strukture važno je za razumijevanje eukariotskih stanica. Sec13 je komponenta NPC čija je uloga u održavanju i stabilizaciji kompleksa nejasna. Njegova dvojna uloga u vezanju proteina Nup145C u sklopu NPC te proteina Sec31 u sklopu vezikula tipa COPII otežava mutacijsku analizu. Cilj ovog projekta bio je riješiti strukturu Sec13 u kompleksu sa insercijskom regijom Nup145C, kako bi se dobila jasna slika veznog mjesta Sec13/Nup145C. Riješena struktura uspoređena je sa strukturama kompleksa Sec13/Sec31. Na temelju razlika među veznim mjestima osmišljen je mutirani Sec13 koji veže isključivo protein Sec31. Posljedica ove mutacije potvrđena je pokusima in vitro.Possession of an envelope-coated nucleus is the defining trait of eukaryotes. Since the nuclear pore complex (NPC) is the only known mediator of nucleo-cytoplasmic traffic, elucidating its structure is important for understanding eukaryotic cells. Sec13 is a component of the NPC whose importance for the stability of the complex is unclear. Its dual role in binding proteins Nup145 in the NPC and Sec31 in COPII vesicles makes mutational analysis of Sec13 function difficult. The goal of this project was solving a structure of Sec13 in complex with the Nup145C insertion blade that would provide a clear view of the Sec13/Nup145C interface. The obtained structure was compared to the structures of the Sec13/Sec31 complex. Based on the differences between the interaction sites of the structures, a mutant Sec13 protein was designed, that binds specifically to Sec31, but has lost affinity for Nup145C. The effect of this mutation was confirmed by in vitro experiments

Active modes of the translocon

All proteins are synthesized in the cytoplasm. However some proteins function outside of cells, in the cell membrane, in the endoplasmic reticulum or one of the connected vesicular systems and need to be sorted and accordingly transported. Consequently, the secretory pathway, a highly conserved protein targeting, translocation and membrane insertion system has evolved to cope with this task. The central actor in the secretory pathway is the heterotrimeric Sec61αβγ protein complex also known as the translocon. This protein complex has been shown to act as a transmembrane channel facilitating protein translocation into the lumen of the endoplasmic reticulum, a starting point in protein secretion. The complex also contains a lateral opening, termed ‘lateral gate’ through which proteins have been suggested to insert into the membrane of the endoplasmic reticulum. The mechanics of translocon opening for protein secretion or membrane insertion has been a matter of debate. The results presented here show secondary structure level resolution cryo-electron microscopic structures of the translocon in complex with stalled ribosome-nascent chain complexes carrying substrates with propensity for either secretion or membrane insertion as well as ribosome-bound inactive translocon molecules. The observed differences in secondary structure suggest a model for translocon activity in protein sorting