Abstract
Band 3 protein is a typical multispanning membrane protein whose membrane topology has been extensively studied from various protein chemical approaches. To clarify the membrane topogenesis of this multispanning protein on the endoplasmic reticulum, the topogenic functions of the anticipated transmembrane segments were individually assessed in an in vitro system using two series of model proteins in which each segment was placed in either a "stop-transfer" context or a "translocation initiation" context. They were expressed in a cell-free system containing rough microsomal membranes, and their topologies were evaluated by taking advantage of either sensitivity to protease or accessibility to N-glycosylation. We found that some segments seem to possess insufficient topogenic functions for membrane integration: the second transmembrane segment (TM2) is insufficient for the stop-transfer sequence, and TM3, TM5, and TM7 are not sufficient for the translocation initiation. In contrast to these phenomena, we herein demonstrate that TM2 shows an efficient stop-transfer function when it is near the preceding TM1 and suggest that TM3, TM5, and TM7 are followed by TM segments with a strong topogenic function to form Nexo/Ccyt topology, via which the preceding segments are integrated into the membrane. From these results, we propose that the interactions between the TMs should be operative during membrane integration, and that the segments with a weak topogenic function are given a transmembrane orientation by their following TMs.
Highlights
Membrane proteins on the secretory pathway in eucaryotic cells are integrated into the membrane at the endoplasmic reticulum (ER)1 and acquire their final membrane topology
According to a hypothesis that has been widely accepted [9, 10], nascent polypeptide on the ribosomes is targeted by the amino-terminal signal sequence in a signal recognition particle-mediated fashion, and the following hydrophobic segments are sequentially integrated into the membrane from the N terminus while showing the alternative functions for either translocation initiation or stop-translocation [11]; after the synthesis of cytoplasmic domain, the following transmembrane segment (TM) initiates the translocation of the following portion, and the transmembrane segment is supposed to stop the ongoing translocation (St; Ref. 9)
We suggest that several TMs in the stop-transfer context possess the potential of internal SA-I, which mediates the insertion of the preceding segment into the membrane
Summary
Membrane proteins on the secretory pathway in eucaryotic cells are integrated into the membrane at the endoplasmic reticulum (ER) and acquire their final membrane topology. According to a hypothesis that has been widely accepted [9, 10], nascent polypeptide on the ribosomes is targeted by the amino-terminal signal sequence in a signal recognition particle-mediated fashion, and the following hydrophobic segments are sequentially integrated into the membrane from the N terminus while showing the alternative functions for either translocation initiation or stop-translocation [11]; after the synthesis of cytoplasmic domain, the following transmembrane segment (TM) initiates the translocation of the following portion (internal SA-II), and the transmembrane segment is supposed to stop the ongoing translocation (St; Ref. 9). The reporter of prolactin was mutated to include the N-glycosylation site by point mutagenesis (T90N)
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