A structural and functional analysis of the docking protein. Characterization of active domains by proteolysis and specific antibodies.
Docking protein is a 73-kDa integral membrane protein of the rough endoplasmic reticulum. It is essential for translocation of nascent secretory proteins into the lumen of the endoplasmic reticulum. Monoclonal and polyclonal antibodies have been generated which, in conjunction with limited proteolysis, have been used to characterize several subspecies of docking protein. These proteolytic fragments have been analyzed with respect to the various functions ascribed to docking protein which can be assayed in vitro. Proteolytic digestion of membrane-associated or of affinity-purified intact docking protein showed that: elastase cleavage generates a 59-kDa soluble fragment and one of 14 kDa which contains the membrane anchoring domain; trypsin as well as endogenous proteolysis generates a 46-kDa fragment, leaving a 27-kDa domain containing the membrane anchor. This 27-kDa fragment can be reduced to a 13- and a 14-kDa piece by elastase digestion. The characteristics of these various subspecies were examined. The 59-kDa soluble fragment, which can reconstitute full translocation activity to docking protein-depleted microsomes (Meyer, D. I., and Dobberstein, B. (1980) J. Cell Biol. 87, 503-508) was capable of releasing a signal recognition particle-mediated translation arrest. The 46-kDa fragment was neither able to reassociate with nor to reconstitute the activity of docking protein-depleted microsomes. Moreover this fragment was unable to release a signal recognition particle-mediated arrest. This suggests that the 13-kDa fragment (the difference between 46 and 59 kDa) is both essential for association with the membrane, and for the release of translation arrests.
Hortsch
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Avossa
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Meyer
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D000818:Animals; D000906:Antibodies; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000032:analysis; D000939:Epitopes / Q000032:analysis; D007425:Intracellular Membranes / Q000032:analysis; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D008861:Microsomes / Q000032:analysis; D008970:Molecular Weight; D010179:Pancreas / Q000032:analysis; D010196:Pancreatic Elastase; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D000906:Antibodies; D000939:Epitopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D010196:Pancreatic Elastase; D014357:Trypsin