Regulation of the ribosome-membrane junction at early stages of presecretory protein translocation in the mammalian endoplasmic reticulum.
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.
Nicchitta
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CV
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA. C.Nicchitta@cellbio.duke.edu
Zheng
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T
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins