Co-translational targeting and translocation of the amino terminus of opsin across the endoplasmic membrane requires GTP but not ATP.
The tight coupling between ongoing translation and translocation across the mammalian endoplasmic reticulum has made it difficult to determine the requirements that are specific for translocation. We have developed an in vitro assay that faithfully mimics the co-translational targeting and translocation of the amino terminus of opsin without ongoing translation. Using this system we demonstrate that this post-translational targeting and translocation requires nucleotide triphosphates but not cytosolic proteins. The addition of GTP alone was sufficient to fully restore targeting. The addition of ATP was not specifically required, and non-hydrolyzable analogs of ATP that blocked 90% of the ATPase activity also had no inhibitory effect on translocation.
Kanner
Elliott M
EM
Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York 10021, USA.
Friedlander
Martin
M
Simon
Sanford M
SM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008322:Mammals; D014176:Protein Biosynthesis; D021381:Protein Transport; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017299:Rod Opsins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate