Yeast ribosomes bind to highly purified reconstituted Sec61 p complex and to mammalian p180

To determine whether the yeast Sec61p translocation pore is a high-affinity ribosome receptor in the endoplasmic reticulum, we isolated the Sec61p com plex using an improved protocol in which contaminants found previously to b e associated with the complex are absent. The purified complex, which conta ins Sec61p with an amino terminal hexahistidine tag, was active since it re scued a sec61-3 post-translational translocation defect in a reconstituted system. Co-reconstitution of the Sec61p and Sec63p complexes into liposomes failed to support post-translational translocation, suggesting that Sec62p is required for this process. By Scatchard analysis, the purified Sec61p c omplex bound to yeast ribosomes when reconstituted into liposomes with a KI D of 5.6nm, a value similar to the KD obtained when ribosome binding to tot al microsomal protein was measured (2.7 nm). In addition, a mammalian prote in, p180, which has been proposed to be a ribosome receptor, was expressed in yeast, and endoplasmic reticulum-derived microsomes isolated from this s train exhibited similar to2.3-fold greater binding to yeast ribosomes. Desp ite this increase in ribosome binding, neither co- nor post-translational t ranslocation was compromised in vivo. In sum, our data suggest that the Sec 61p complex is a ribosome receptor in the yeast endoplasmic reticulum membr ane.