An in vitro assay using overexpressed yeast SRP demonstrates that cotranslational translocation is dependent upon the J-domain of Sec63p.
The signal recognition particle (SRP) is required for co-translational targeting of polypeptides to the endoplasmic reticulum (ER). Once at the membrane, the precursor interacts with a complex proteinaceous machinery that mediates its translocation across the bilayer. Genetic studies in yeast have identified a number of genes whose products are involved in this complex process. These mutants offer a potentially valuable resource with which to analyze the biochemical role played by each component in the pathway. However, such analyses have been hampered by the failure to reconstitute an efficient in vitro assay for SRP-dependent translocation. We report the construction of two multicopy vectors that allow overexpression of all seven gene products required to make SRP in the yeast Saccharomyces cerevisiae. The overexpressed subunits assemble into intact and functional SRP particles, and we further demonstrate that in vitro reconstitution of co-translational translocation is greatly enhanced using cytosol from the overexpression strain. We use this assay to demonstrate that Sec63p is required for co-translational translocation in vitro and specifically identify the "J-domain" of Sec63p as crucial for this pathway.
Willer
Martin
M
School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Jermy
Andrew J
AJ
Steel
Gregor J
GJ
Garside
Helen J
HJ
Carter
Stephanie
S
Stirling
Colin J
CJ
D001483:Base Sequence; D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion / Q000502:physiology; D018271:Signal Recognition Particle / Q000096:biosynthesis / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D006360:Heat-Shock Proteins; D026901:Membrane Transport Proteins; D009841:Oligonucleotides; C074215:SEC63 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle