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 complex (translocon) in the rough endoplasmic reticulum (rough ER) membrane. Translating or nontranslating ribosomes bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present evidence that the beta subunit of the complex (Sec61beta) makes contact with nontranslating ribosomes. A fusion protein containing the Sec61beta cytoplasmic domain (Sec61beta(c)) prevents the binding of ribosomes to stripped ER-derived membranes and also binds to ribosomes directly with an affinity close to the affinity of ribosomes for stripped ER-derived membranes. The ribosome binding activity of Sec61beta(c), like that of native ER membranes, is sensitive to high salt concentrations and is not based on an unspecific charge-dependent interaction of the relatively basic Sec61beta(c) domain with ribosomal RNA. Like stripped ER membranes, the Sec61beta(c) sequence binds to large ribosomal subunits in preference over small subunits. Previous studies have shown that Sec61beta is inessential for ribosome binding and protein translocation, but translocation is impaired by the absence of Sec61beta, and it has been proposed that Sec61beta assists in the insertion of nascent proteins into the translocation pore. Our results suggest a physical interaction of the ribosome itself with Sec61beta; 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.
Levy
R
R
Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.
Wiedmann
M
M
Kreibich
G
G
D000818:Animals; D001667:Binding, Competitive; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D021381:Protein Transport; D012343:RNA, Transfer / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D000069816:SEC Translocation Channels
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D012343:RNA, Transfer; D005982:Glutathione Transferase