Biochemical fractionation and assembly of the membrane components that mediate nascent chain targeting and translocation.
Fractionation of a microsomal detergent extract with ammonium sulfate allows separation of the signal recognition particle receptor (SR alpha), which is required for targeting of the nascent chain, from other microsomal proteins, such as signal peptidase, whose activity is displayed during subsequent translocation. The reconstituted SR alpha-enriched fraction is functional in assays of precursor targeting and elongation arrest release but lacks translocation activity. This defect can be complemented by addition, prior to reconstitution, of a separate protein subfraction. In addition, protein components necessary for translocation can be reversibly depleted from the complementary fraction, under conditions where precursor targeting is retained, by sulfhydryl-directed chromatography. Thus, precursor binding and translocation can be biochemically uncoupled, indicating that they are sequential reactions mediated by distinct components.
Nicchitta
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Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021-6399.
Migliaccio
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Blobel
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D000818:Animals; D002458:Cell Fractionation; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D011388:Prolactin / Q000235:genetics; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D012156:Reticulocytes / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D018271:Signal Recognition Particle; D014908:Triticum / Q000378:metabolism
D008565:Membrane Proteins; D011498:Protein Precursors; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle; C036318:signal peptide receptor; C017671:preprolactin; D011388:Prolactin