Yeast ribosomes bind to highly purified reconstituted Sec61p 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 complex using an improved protocol in which contaminants found previously to be associated with the complex are absent. The purified complex, which contains Sec61p with an amino terminal hexahistidine tag, was active since it rescued 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 complex bound to yeast ribosomes when reconstituted into liposomes with a KD of 5.6 nM, a value similar to the KD obtained when ribosome binding to total microsomal protein was measured (2.7 nM). In addition, a mammalian protein, p180, which has been proposed to be a ribosome receptor, was expressed in yeast, and endoplasmic reticulum-derived microsomes isolated from this strain exhibited approximately 2.3-fold greater binding to yeast ribosomes. Despite this increase in ribosome binding, neither co- nor post-translational translocation was compromised in vivo. In sum, our data suggest that the Sec61p complex is a ribosome receptor in the yeast endoplasmic reticulum membrane.
Morrow
M W
MW
Department of Biological Sciences, University of Pittsburgh, 267 Crawford Hall, Pittsburgh, PA 15260, USA.
Brodsky
J L
JL
D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006360:Heat-Shock Proteins; D008322:Mammals / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D000069816:SEC Translocation Channels; D029701:Saccharomyces cerevisiae Proteins; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D020648:Peptide Elongation Factor 1; D018160:Receptors, Cytoplasmic and Nuclear; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; C074215:SEC63 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C065526:ribosome receptor