In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide–associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.
Spontaneous Release of Cytosolic Proteins from Posttranslational Substrates before Their Transport into the Endoplasmic Reticulum
Dr. Plath's present address is Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.
Abbreviations used in this paper: CCT, chaperonin containing TCP1; IP, immunoprecipitation; NAC, nascent polypeptide–associated complex; p60/Hop, Hsp70/Hsp90 organizing protein; ppαF, prepro-α factor; RNC, ribosome–nascent chain complex; SRP, signal recognition particle; TCP1, tailless complex polypeptide; TRiC, TCP1–ring complex; wt, wild-type.
Kathrin Plath, Tom A. Rapoport; Spontaneous Release of Cytosolic Proteins from Posttranslational Substrates before Their Transport into the Endoplasmic Reticulum. J Cell Biol 2 October 2000; 151 (1): 167–178. doi: https://doi.org/10.1083/jcb.151.1.167
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