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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome-membrane
junction in the mammalian endoplasmic reticulum. As
a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation
products were used to delimit the analysis to defined
stages of translocation.
In a study of secretory protein precursors, formation
of a protease-resistant ribosome-membrane junction,
currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and
stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to
the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined
as a function of the stage of translocation, and was
found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic
face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound
intermediates did not disrupt subsequent translocation,
suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound
ribosome. A model describing a potential mode of regulation of the ribosome-membrane junction by the nascent chain is presented.
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