Abstract
Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.
Highlights
Ribosomes translate the linear genetic code into polypeptide chains that must fold into a specific three-dimensional structure and often assemble with other polypeptides to be born as functional proteins
Molecular midwives called chaperones often bind to nascent protein chains as they emerge from the ribosome to help them fold
Sucrose gradient fractionation confirmed that the TAP-tagged Srp54, all three Nascent Chain Associated Complex (NAC) subunits, and Rpl16 and Rpl17 all associated with polysomes (Figure S1A and Figure S6B)
Summary
Ribosomes translate the linear genetic code into polypeptide chains that must fold into a specific three-dimensional structure and often assemble with other polypeptides to be born as functional proteins. During this process, as nascent proteins emerge from the ribosome, they lack information to complete their folding and are susceptible to misfolding and aggregation. A diverse set of molecular chaperones act as midwives to stabilize and facilitate the folding of newly translated polypeptides into functional proteins. A number of specific targeting factors recognize nascent polypeptides before they have a chance to fold in the cytosol and deliver them to specific cellular membranes. One of the best understood mechanisms involves the cotranslational recognition of characteristic hydrophobic nascent chain segments by the Signal Recognition Particle (SRP), which facilitates proper delivery of the entire ribosome-nascent chain complex (RNC) to the ER membrane for cotranslational translocation
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