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. Among these, Chaperones Linked to Protein Synthesis (CLIPS) 
interact physically with ribosomes and associate cotranslationally with nascent polypeptides. In addition to folding within the cytosol, many polypeptides must be directed to various membrane-bound organelles, such as the ER and mitochondria. 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.
The multiplicity of fates and possible interactions available to a polypeptide as it emerges from the ribosome in the eukaryotic cytosol raises a number of intriguing questions. Do all nascent chains interact with chaperones? Is there any specificity in the recognition of nascent chains by chaperones? How do cytosolic chaperones and targeting factors such as SRP discriminate among their respective substrates, and how is the fidelity of this process achieved? These questions are fundamental to understanding the mechanisms governing polypeptide fate as it emerges from the ribosome.
Much of our understanding of nascent chain interactions with chaperones or other targeting factors comes from the study of model proteins, chosen for a convenient enzymatic or structural assay for folding or translocation. As a result, the overall logic and organization of the system that mediates the critical events in delivery and birth of a nascent polypeptide as a functional protein is still a mystery. To begin to fill this gap, we developed a systematic approach to define the principles underlying the specificity of cotranslational chaperones. In the present work, we apply it to study the specificity and interplay of two important nascent-chain interacting factors: SRP and the Nascent Chain Associated Complex (NAC).
Eukaryotic SRP was initially identified as a factor for targeting proteins to the ER. SRP is a ribonucleoprotein complex comprising six proteins (in yeast Srp72, Srp68, Srp54, Sec65, Srp21, Srp14) and a non-coding RNA (scR1) 
. SRP binds weakly to all ribosomes, even those that do not translate ER-destined proteins, by virtue of its contacts with multiple ribosomal sites. One of the contact sites, the ribosomal protein Rpl25, is also a proposed binding site for NAC 
. SRP recognizes characteristic hydrophobic sequences such as the N-terminal signal sequence (SS) and transmembrane domains (TM) in nascent polypeptides as they emerge from the ribosome. The dual recognition of ribosome and nascent chain by SRP ensures high affinity for cognate RNCs. SRP-bound RNCs are targeted to the membrane through interaction with the membrane bound SRP receptor (SR), where nascent chains bearing SS or TM domains are translocated across the ER membrane by a protein complex called the Sec61 translocon. Interestingly, the Sec61 translocon itself can also interact directly with ribosomes 
, preferentially recognizing RNCs bearing hydrophobic SS or TM regions; this might provide an SRP independent route to the ER or a proofreading mechanism for ER import. SRP-independent co- and post-translational ER targeting pathways also exist, including post-translational chaperone-assisted translocation 
and direct ER targeting of mRNA through RNA-binding proteins (reviewed in 
). The respective contributions of the various targeting pathways to ER import in vivo and the determinants that channel an ER-bound protein through either SRP-dependent or SRP-independent pathways are not entirely understood.
Very little is known about the function of the abundant and ubiquitous NAC complex. NAC is phylogenetically conserved across eukaryotes and archaea but is absent from prokaryotes 
. Structural characterization of archaeal NAC indicates that its subunits must assemble in tightly folded dimers 
. Most NAC complexes are heterodimers of two subunits, α and β, but homodimers have also been reported 
. Yeast contains a single alpha subunit gene, EGD2
, and two β subunit genes, EGD1
. NAC contacts Rpl25 
and Rpl31 
in close proximity to the ribosomal exit site and can crosslink to very short nascent chains 
, suggesting an early role in the birth of nascent proteins. NAC deletion causes embryonic lethality in mice, flies, and nematodes 
but only minor growth defects in yeast 
. Despite its abundance and conservation, the specificity and function of NAC are obscure and controversial. NAC does not associate with proteins after release from the ribosome and has no apparent chaperone activity. From in vitro experiments, NAC was initially proposed to be essential for faithful SRP-targeting of proteins to the ER 
and preventing inappropriate association of RNCs lacking SS or TM with the translocon Sec61 
. This hypothesis was not supported, however, by subsequent in vitro and in vivo studies, which did not reveal aberrant translocation phenotypes in NAC-deleted strains 
. A regulatory role for NAC in mitochondrial protein import, suggested by in vitro experiments 
, was not corroborated by in vivo studies 
. Given the robustness of protein homeostasis pathways, loss of NAC could be compensated by other systems. Indeed, NAC deletions exacerbate the effect of deleting the yeast Hsp70 homolog SSB
, leading to higher levels of ribosomal protein aggregation 
A number of experimental challenges have hindered progress towards understanding the robust network of chaperones and cofactors acting cotranslationally on nascent chains. Because nascent chains comprise a small, transient, and heterogeneous cellular pool of chaperone substrates, proteomic analyses are currently impractical. The high degree of redundancy within the cellular chaperone network often masks obvious loss-of-function phenotypes. Our understanding of the specificity and mechanism of cotranslationally acting chaperones comes from in vitro translation experiments using individual model proteins, and thus the generality of such experiments is hard to ascertain. To circumvent these difficulties, we developed a sensitive, systematic method for defining the substrate specificity and interplay of cotranslationally acting chaperones and other nascent chain binding and modifying factors (e.g., acetylation enzymes) in vivo. Here we used this approach to characterize the specificity of the interactions of SRP and NAC with nascent polypeptides and how the interplay between these two factors serves to modulate that specificity.