NAC guides a ribosomal multienzyme complex for nascent protein processing.

Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood.

Cotranslational sorting and processing of newly synthesized proteins in eukaryotes.

Ribosomes interact with a variety of different protein biogenesis factors that guide newly synthesized proteins to their native 3D shapes and cellular localization. Depending on the type of translated substrate, a distinct set of cotranslational factors must interact with the ribosome in a timely and coordinated manner to ensure proper protein biogenesis. While cytonuclear proteins require cotranslational maturation and folding factors, secretory proteins must be maintained in an unfolded state and processed cotranslationally by transport and membrane translocation factors.

NAC controls cotranslational N-terminal methionine excision in eukaryotes.

N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1.

Molecular basis of the TRAP complex function in ER protein biogenesis.

The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane.

Mechanism of signal sequence handover from NAC to SRP on ribosomes during ER-protein targeting.

The nascent polypeptide-associated complex (NAC) interacts with newly synthesized proteins at the ribosomal tunnel exit and competes with the signal recognition particle (SRP) to prevent mistargeting of cytosolic and mitochondrial polypeptides to the endoplasmic reticulum (ER). How NAC antagonizes SRP and how this is overcome by ER targeting signals are unknown. Here, we found that NAC uses two domains with opposing effects to control SRP access.

Deciphering molecular details of the RAC-ribosome interaction by EPR spectroscopy.

The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC-ribosome contact affecting translational accuracy.

Snapshots of native pre-50S ribosomes reveal a biogenesis factor network and evolutionary specialization.

Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit.

Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC.

Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone.

Dual Role of Ribosome-Binding Domain of NAC as a Potent Suppressor of Protein Aggregation and Aging-Related Proteinopathies.

The nascent polypeptide-associated complex (NAC) is a conserved ribosome-associated protein biogenesis factor. Whether NAC exerts chaperone activity and whether this function is restricted to de novo protein synthesis is unknown. Here, we demonstrate that NAC directly exerts chaperone activity toward structurally diverse model substrates including polyglutamine (PolyQ) proteins, firefly luciferase, and Aβ40.

Chaperone Interactions at the Ribosome.

The continuous refreshment of the proteome is critical to maintain protein homeostasis and to adapt cells to changing conditions. Thus, de novo protein biogenesis by ribosomes is vitally important to every cellular system. This process is delicate and error-prone and requires, besides cytosolic chaperones, the guidance by a specialized set of molecular chaperones that bind transiently to the translation machinery and the nascent protein to support early folding events and to regulate cotranslational protein transport.

Neomycin-dependent hammerhead ribozymes for the direct control of gene expression in Saccharomyces cerevisiae.

Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae.

Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths.

Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states.

Conformational flexibility within the nascent polypeptide-associated complex enables its interactions with structurally diverse client proteins.

As newly synthesized polypeptides emerge from the ribosome, it is crucial that they fold correctly. To prevent premature aggregation, nascent chains interact with chaperones that facilitate folding or prevent misfolding until protein synthesis is complete. Nascent polypeptide-associated complex (NAC) is a ribosome-associated chaperone that is important for protein homeostasis.

Vms1: A Cytosolic CAT-Tailing Antagonist to Protect Mitochondria.

In eukaryotes, a cytosolic ribosome quality control complex recycles erroneously stalled ribosomes and modifies faulty nascent chains by ubiquitination and by C-terminal Ala- and Thr-extension (CAT-tailing). Reported recently in Cell, Izawa et al. identify cytosolic Vms1 (VCP/Cdc48-associated mitochondrial stress-responsive 1) as an inhibitor of CAT-tailing, which prevents mitochondrial dysfunction caused by imported CAT-tailed polypeptides.

Multivalent contacts of the Hsp70 Ssb contribute to its architecture on ribosomes and nascent chain interaction.

Hsp70 chaperones assist de novo folding of newly synthesized proteins in all cells. In yeast, the specialized Hsp70 Ssb directly binds to ribosomes. The structural basis and functional mode of recruitment of Ssb to ribosomes is not understood.

Ribosome Assembly as Antimicrobial Target.

Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly.

Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae.

Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β'-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ'-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells.

Not4-dependent translational repression is important for cellular protein homeostasis in yeast.

Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis.

A comparison of the folding characteristics of free and ribosome-tethered polypeptide chains using limited proteolysis and mass spectrometry.

The kinetics and thermodynamics of protein folding are commonly studied in vitro by denaturing/renaturing intact protein sequences. How these folding mechanisms relate to de novo folding that occurs as the nascent polypeptide emerges from the ribosome is much less well understood. Here, we have employed limited proteolysis followed by mass spectrometry analyses to compare directly free and ribosome-tethered polypeptide chains of the Src-homology 3 (SH3) domain and its unfolded variant, SH3-m10.

Fluorescence-based monitoring of ribosome assembly landscapes.

Background: Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control.

Results: We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.

The principle of antagonism ensures protein targeting specificity at the endoplasmic reticulum.

The sorting of proteins to the appropriate compartment is one of the most fundamental cellular processes. We found that in the model organism Caenorhabditis elegans, correct cotranslational endoplasmic reticulum (ER) transport required the suppressor activity of the nascent polypeptide-associated complex (NAC). NAC did not affect the accurate targeting of ribosomes to ER translocons mediated by the signal recognition particle (SRP) pathway but inhibited additional unspecific contacts between ribosomes and translocons by blocking their autonomous binding affinity.

Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli.

While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain--using chromosomal gene knock-in techniques--that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively.

The nascent polypeptide-associated complex is a key regulator of proteostasis.

The adaptation of protein synthesis to environmental and physiological challenges is essential for cell viability. Here, we show that translation is tightly linked to the protein-folding environment of the cell through the functional properties of the ribosome bound chaperone NAC (nascent polypeptide-associated complex). Under non-stress conditions, NAC associates with ribosomes to promote translation and protein folding.

Macrolides: the plug is out.

Macrolide antibiotics are thought to clog up the ribosomal tunnel and thereby block general protein synthesis. By using a combination of elegant in vivo and in vitro approaches, Kannan et al. show that the inhibitory action of these drugs on bacterial protein synthesis is selective rather than global.