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. The core globular domain prevented SRP from binding to signal-less ribosomes, whereas a flexibly attached domain transiently captured SRP to permit scanning of nascent chains. The emergence of an ER-targeting signal destabilized NAC's globular domain and facilitated SRP access to the nascent chain. These findings elucidate how NAC hands over the signal sequence to SRP and imparts specificity of protein localization.

Receptor compaction and GTPase rearrangement drive SRP-mediated cotranslational protein translocation into the ER.

The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)-driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases.

Fidelity of Cotranslational Protein Targeting to the Endoplasmic Reticulum.

Fidelity of protein targeting is essential for the proper biogenesis and functioning of organelles. Unlike replication, transcription and translation processes, in which multiple mechanisms to recognize and reject noncognate substrates are established in energetic and molecular detail, the mechanisms by which cells achieve a high fidelity in protein localization remain incompletely understood. Signal recognition particle (SRP), a conserved pathway to mediate the localization of membrane and secretory proteins to the appropriate cellular membrane, provides a paradigm to understand the molecular basis of protein localization in the cell. In this chapter, we review recent progress in deciphering the molecular mechanisms and substrate selection of the mammalian SRP pathway, with an emphasis on the key role of the cotranslational chaperone NAC in preventing protein mistargeting to the ER and in ensuring the organelle specificity of protein localization.

A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex.

Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.

Timing and specificity of cotranslational nascent protein modification in bacteria.

The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.

Aminophenylboronic acid polymer nanoparticles for quantitation of glucose and for insulin release.

We have developed a simple route for the preparation of aminophenylboronic acid polymer nanoparticles (APB PNs) from 3-aminophenylboronic acid and formaldehyde under alkaline conditions according to an extended StÓ§ber method. Insulin and R6G have been selected to prepare functional insulin-APB PNs and R6G-APB PNs, respectively. During the formation of APB PNs, the representative molecules are embedded inside the APB PNs. Through specific binding of glucose with boronic acid moieties on the R6G-APB PNs and insulin-APB PNs, glucose induces expansion of the APB PNs, leading to release of R6G and insulin molecules, respectively. As a result of release of R6G molecules, the fluorescence intensity of R6G-APB PN solution increases, allowing quantitation of glucose in PBS solutions (10 mM, pH 7.4) with a linear range over 0-10 mM. Release of insulin from insulin-APB PNs is significant and rapid when the glucose concentration is higher than 7 mM. Having advantages of low cost, simple preparation, biocompatibility, and continuous response to glucose, the insulin-APB PNs hold great potential as an alternative for treating diabetic patients. Graphical Abstract Quantitation of glucose and release of insulin by glucose responsive 3-aminophenylboronic acid polymer nanoparticles.

Aptamer-conjugated polymeric nanoparticles for the detection of cancer cells through "turn-on" retro-self-quenched fluorescence.

We have developed a simple, sensitive, and rapid fluorescence assay for the detection of cancer cells, based on "turn-on" retro-self-quenched fluorescence inside the cells. 1,3-Phenylenediamine resin (DAR) nanoparticles (NPs) containing rhodamine 6G (R6G) are conjugated with aptamer (apt) sgc8c to prepare sgc8c-R6GDAR NPs, while that containing rhodamine 101 (R101) are conjugated with TD05 for the preparation of TD05-R101DAR NPs. The sgc8c-R6GDAR and TD05-R101DAR NPs separately recognize CCRF-CEM and Ramos cells. The fluorescence intensities of the two apt-DAR NPs are both weak due to self-quenching, but they increase inside the cells as a result of release of the fluorophores from the apt-DAR NPs. The apt-DAR NPs' structure becomes less compact at low pH value, leading to the release of the fluorophores. The sgc8c-R6GDAR and TD05-R101DAR NPs allow detection of as low as 44 CCRF-CEM cells and 79 Ramos cells mL(-1), respectively, using a commercial reader within 10 min. Practicality of the two probes have been validated by the quantitation and identification of CCRF-CEM and Ramos cells spiked in blood samples through conventional fluorescence and flow cytometry analysis, with advantages of sensitivity, selectivity, and rapidity.