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Dynamic insertion of membrane proteins at the endoplasmic reticulum

Higy, Marie. Dynamic insertion of membrane proteins at the endoplasmic reticulum. 2005, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7180

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Abstract

Most eukaryotic membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane by the Sec61 translocation complex. They are targeted to the translocon by hydrophobic signal sequences which induce the translocation of either their N- or C-terminal sequence. Signal sequence orientation is largely determined by charged residues flanking the apolar sequence (the positive-inside rule), folding properties of the N-terminal segment, and the hydrophobicity of the signal. Recent in vivo experiments suggest that N-terminal signals initially insert into the translocon head-on to yield a translocated Nterminus. Driven by a local electrical potential, the signal may invert its orientation and translocate the C-terminal sequence. Increased hydrophobicity slows down inversion by stabilizing the initial bound state. In vitro crosslinking studies indicate that signals rapidly contact lipids upon entering the translocon. Together with the recent crystal structure of the homologous SecYEβ translocation complex of Methanococcus jannaschii, which did not reveal an obvious hydrophobic binding site for signals within the pore, a model emerges in which the translocon allows the lateral partitioning of hydrophobic segments between the aqueous pore and the lipid membrane. Signals may return into the pore for reorientation until translation is terminated. Subsequent transmembrane segments in multispanning proteins behave similarly and contribute to the overall topology of the protein. This thesis was aimed at investigating the integration of single- and doublespanning membrane proteins in mammalian cells. The first part consisted of probing the environment of the signal while its orientation is determined by inserting different hydrophobic residues at various positions throughout a uniform oligo-leucine signal sequence. The resulting topologies revealed a strikingly symmetric position dependence specifically for bulky aromatic amino acids, reflecting the structure of a lipid bilayer. The results support the model that during topogenesis in vivo the signal sequence is exposed to the lipid membrane. The second part consisted of the determination of the kinetics of double-spanning protein topogenesis. The results confirmed that major reorientation of the polypeptide my occur when a second topogenic sequence, conflicting with a first one, enters the translocon. They also showed that the time window for protein reorientation differs for different types of substrate.
Advisors:Spiess, Martin
Committee Members:Hauri, Hans-Peter
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biochemistry (Spiess)
UniBasel Contributors:Spiess, Martin and Hauri, Hans-Peter
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7180
Thesis status:Complete
Number of Pages:91
Language:English
Identification Number:
edoc DOI:
Last Modified:02 Aug 2021 15:04
Deposited On:13 Feb 2009 15:12

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