Abstract
The reinitiation of the translocation of the growing nascent chain across the endoplasmic reticulum membrane is essential for the topogenesis of multispanning membrane proteins. We investigated the requirements for the reinitiation process using model proteins in which systematically designed sequences were inserted after two preceding topogenic sequences, namely the N-terminal signal sequence (S) and stop transfer sequence (St). The model proteins were translated in vitro in the presence of rough microsomes, and the final topology of the proteins in the microsomal membrane was examined by proteolytic digestion. The structural requirements for S and the reinitiation sequence (R) overlapped to some extent, but substantial differences were noticed. When St and R were separated by a short cytoplasmic segment (58 amino acids), the efficiency of the reinitiation was not affected by the concentration of the signal recognition particle (SRP) in the translation system, even though the sequence inserted as R was an SRP-dependent signal sequence. However, when the cytoplasmic segment was longer (100 amino acids), the reinitiation efficiency was reduced, and the SRP improved the overall efficiency as well as impaired the accessibility of the processing site after the R to the signal peptidase.
Highlights
In eucaryotic cells, membrane proteins on the endocytotic and exocytotic pathway are co-translationally integrated into the endoplasmic reticulum membrane, and their final membrane topologies are defined during their integration
They are targeted to the endoplasmic reticulum membrane by the signal recognition particle (SRP)1 and SRP-receptor system [1], and they are inserted into the endoplasmic reticulum membrane depending on the hydrophilic protein translocation channel, which consists of several protein subunits [2, 3]
Constructed model sequences were fused to the N terminus of mature interleukin 2 (IL2) and examined for their ability as S (S context) in the previous study [12]
Summary
Membrane proteins on the endocytotic and exocytotic pathway are co-translationally integrated into the endoplasmic reticulum membrane, and their final membrane topologies are defined during their integration. Both types of SAs are recognized and targeted to the membrane by SRP [9] The functions of these topogenic sequences are mainly determined by the hydrophobic segment and are modulated by adjacent charged, and mainly positive, amino acid residues. It has been demonstrated that the membrane integration of proteins is a sequential event of the insertion of the alternative hydrophobic sequences starting from the Nterminal transmembrane segment and that the hydrophobic segment, which is not recognizable by the SRP, can mediate the second translocation [17] It is, not clear whether the structural requirements for R are similar to those of S and whether some proteinaceous factors, such as the SRP and/or others, participate in the process.
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