In vitro binding of ribosomes to the beta subunit of the Sec61p protein translocation complex

The Sec61p complex forms the core element of the protein translocation comp lex (translocon) in the rough endoplasmic reticulum trough ER) membrane. Tr anslating or nontranslating ribosomes bind with high affinity to ER membran es that have been stripped of ribosomes or to liposomes containing purified Sec61p, Here we present evidence that the beta subunit of the complex (Sec 61 beta) makes contact with nontranslating ribosomes, A fusion protein cont aining the Sec61 beta cytoplasmic domain (Sec61 beta (c)) prevents the bind ing of ribosomes to stripped ER-derived membranes and also binds to ribosom es directly with an affinity close to the affinity of ribosomes for strippe d ER-derived membranes. The ribosome binding activity of Sec61 beta (c), li ke that of native ER membranes, is sensitive to high salt concentrations an d is not based on an unspecific charge-dependent interaction of the relativ ely basic Sec61 beta (c) domain with ribosomal RNA. Like stripped ER membra nes, the Sec61 beta (c) sequence binds to large ribosomal subunits in prefe rence over small subunits, Previous studies have shown that Sec61 beta is i nessential for ribosome binding and protein translocation, but translocatio n is impaired by the absence of Sec61 beta (c) and it has been proposed tha t Sec61 beta assists in the insertion of nascent proteins into the transloc ation pore. Our results suggest a physical interaction of the ribosome itse lf with Sec61 beta; this may normally occur alongside interactions between the ribosome and other elements of Sec61p, or it may represent one stage in a temporal sequence of binding.