Formation of a functional ribosome-membrane junction during translocation requires the participation of a GTP-binding protein.
The requirement for ribonucleotides and ribonucleotide hydrolysis was examined at several distinct points during translocation of a secretory protein across the endoplasmic reticulum. We monitored binding of in vitro-assembled polysomes to microsomal membranes after removal of ATP and GTP. Ribonucleotides were not required for the initial low salt-insensitive attachment of the ribosome to the membrane. However, without ribonucleotides the nascent secretory chains were sensitive to protease digestion and were readily extracted from the membrane with either EDTA or 0.5 M KOAc. In contrast, nascent chains resisted extraction with either EDTA or 0.5 M KOAc and were insensitive to protease digestion after addition of GTP or nonhydrolyzable GTP analogues. Translocation of the nascent secretory polypeptide was detected only when ribosome binding was conducted in the presence of GTP. Thus, translocation-competent binding of the ribosome to the membrane requires the participation of a novel GTP-binding protein in addition to the signal recognition particle and the signal recognition particle receptor. The second event we examined was translocation and processing of a truncated secretory polypeptide. Membrane-bound polysomes bearing an 86-residue nascent chain were generated by translation of a truncated preprolactin mRNA. Ribonucleotide-independent translocation of the polypeptide was detected by cleavage of the 30-residue signal sequence after puromycin termination. Nascent chain transport, per se, is apparently dependent upon neither ribonucleotide hydrolysis nor continued elongation of the polypeptide once a functional ribosome-membrane junction has been established.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004285:Dogs; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure*; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure*; D010179:Pancreas / Q000378:metabolism / Q000648:ultrastructure; D014176:Protein Biosynthesis / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure*
D011691:Puromycin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins